Method to Identify Disease Resistant Quantitative Trait Loci in Soybean and Compositions Thereof

ABSTRACT

The present invention is in the field of plant breeding and genetics, particularly as it pertains to the genus,  Glycine . More specifically, the invention relates to a method for screening soybean plants containing one or more quantitative trait loci for disease resistance, species of  Glycine  having such loci and methods for breeding for and screening of  Glycine  with such loci. The invention further relates to the use of exotic germplasm in a breeding program.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/738,197 filed on Jun. 12, 2015, which is a continuation of U.S.patent application Ser. No. 14/191,044 filed on Feb. 26, 2014 (U.S. Pat.No. 9,091,681), which is a continuation of U.S. patent application Ser.No. 13/529,851 filed on Jun. 21, 2012 (U.S. Pat. No. 8,692,054), whichis a continuation of U.S. patent application Ser. No. 13/178,668 filedon Jul. 8, 2011 (U.S. Pat. No. 8,389,798). U.S. patent application Ser.No. 13/178,668 is a continuation of U.S. patent application Ser. No.12/472,906 filed on May 27, 2009 (U.S. Pat. No. 7,994,395), which is adivisional of U.S. patent application Ser. No. 11/805,667 filed on May24, 2007 (U.S. Pat. No. 7,951,998), which claims the benefit of priorityto U.S. Provisional Application Ser. No. 60/808,430 filed May 25, 2006(expired). Each of the aforementioned applications is herebyincorporated by reference it is entirety.

INCORPORATION OF THE SEQUENCE LISTING

This application contains a sequence listing, submitted herewithelectronically via EFS web, containing the file named“P30876US07_Seq.txt” which is 71,615 bytes in size (measured in Windows7), which was created on May 8, 2018, and which is herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of plant breeding and diseaseresistance. More specifically, the invention relates to a method forscreening plants from the genus Glycine containing quantitative traitloci that are associated with disease resistance and methods forbreeding disease resistant Glycine plants. The disease can be caused bya fungus, virus, bacterium, or invertebrate animal. The inventionfurther relates to the use of accession germplasm containingquantitative trait loci (QTL) conferring disease resistance forintrogression into elite germplasm in a breeding program for resistanceto the fungal pathogen, Phakopsora pachyrhizi.

BACKGROUND OF THE INVENTION

The soybean, Glycine max (L.) Merril, is one of the major economic cropsgrown worldwide as a primary source of vegetable oil and protein(Sinclair and Backman, Compendium of Soybean Diseases, 3^(rd) Ed. APSPress, St. Paul, Minn., p. 106. (1989)). The growing demand for lowcholesterol and high fiber diets has also increased soybean's importanceas a health food.

Soybean yields in the United States are reduced each year by diseases.High yields per hectare are critical to a farmer's profit margin,especially during periods of low prices for soybean. The financial losscaused by soybean diseases is important to rural economies and to theeconomies of allied industries in urban areas. The effects of theselosses are eventually felt throughout the soybean market worldwide.Estimates of loss due to disease in the United States and Ontario varyfrom year to year and by disease. From 1999 to 2002 soybean yield lossestimates were in the range of 8 million metric tons to 10 millionmetric tons in the United States and 90,000 to 166,000 metric tons inOntario (Wrather et al., Online. Plant Health Progressdoi:10:1094/PHP-2003-0325-01-RV).

Asian Soybean Rust (herein referred to as ASR) has been reported in theEastern and Western Hemispheres. In the Eastern Hemisphere, ASR has beenreported in Australia, China, India, Japan, Taiwan and Thailand. In theWestern Hemisphere, ASR has been observed in Brazil, Columbia, CostaRica and Puerto Rico. ASR can be a devastating disease, causing yieldlosses of up to 70 to 80% as reported in some fields in Taiwan. Plantsthat are heavily infected have fewer pods and smaller seeds that are ofpoor quality (Frederick et al., Mycology 92: 217-227 (2002)). ASR wasfirst observed in the United States in Hawaii in 1994. ASR was laterintroduced into the continental United States in the fall of 2004,presumably as a consequence of tropical storm activity. Modelpredictions indicated that ASR had been widely dispersed throughout thesoutheastern United States, and subsequent field and laboratoryobservations confirmed this distribution.

Two species of fungi, Phakopsora pachyrhizi Sydow and Phakopsorameibomiae (Arthur) Arthur, cause ASR. Unlike other rusts, P. pachyrhiziand P. meibomiae infect an unusually broad range of plant species. P.pachyrhizi is known to naturally infect 31 species in 17 genera oflegumes and 60 species in 26 other genera have been infected undercontrolled conditions. P. meibomiae naturally infects 42 species in 19genera of legumes, and 18 additional species in 12 other genera havebeen artificially infected. Twenty-four plant species in 19 genera arehosts for both species (Frederick et al., Mycology 92: 217-227 (2002)).

Soybean plants resistant to ASR have been identified. Four dominant,independently inherited race-specific QTL for resistance to P.pachyrhizi, herein designated ASR resistance locus 1, ASR resistancelocus 2, ASR resistance locus 3, and ASR resistance locus 4, have beenidentified in PI 200492, PI 230970, PI 462312 (Ankur), and PI 459025B,respectively. These lines, as well as seven others, are suspected ofcontaining QTL for ASR resistance. PI 239871A and PI 239871B (G. soja),PI 230971 and PI 459024B, and the cultivars Taita Kaohsiung-5,Tainung-4, and Wayne have been used as differentials to identify nineraces at the Asian Vegetable Research and Development Center, in Taiwan.The predominant race was compatible with three or more of thedifferentials, indicating that some races already possess multiplevirulence factors to known and suspected genes for resistance.Resistance also occurs among the wild Glycine spp. from Australia.Rate-reducing resistance has also been demonstrated. However, it isdifficult to evaluate this type of resistance because the rate of rustdevelopment is dependent on soybean development and maturity (Sinclairet al., eds., Soybean rust workshop. College of Agricultural, Consumer,and Environmental Sciences. Natl. Soybean Res. Lab. Publ. 1 (1996)).

Evaluating plants that could potentially contain QTL conferringresistance to ASR can be time consuming and require large amounts ofbiologically contained space. Culturing P. pachyrhizi requires the useof an approved biological containment hood. In addition, greenhouses andgrowth chambers used to grow plants for ASR resistance testing will haveto be constructed in a manner that prevents the accidental release ofthe organism, especially in locations in which the organism has stillnot yet been observed. Different cultures of P. pachyrhizi may possessdifferent virulence factors. Over time, new strains of P. pachyrhizi maybe introduced into the United States. Therefore, any breeding programdesigned to breed resistance into soybean against ASR will need to beable to respond rapidly to changes in the P. pachyrhizi population.Also, breeding for soybean crops used in other geographic locations willrequire selecting resistance to the specific strains that affect thoseregions, in addition to providing those agronomic characteristics thatare preferred by these farmers in that region. Therefore, there is agreat need for a rapid, time and cost efficient high throughput methodfor screening germplasm resistant to ASR. This method must not onlyprovide speed and efficiency, but must also be able to be performed witha minimal amount of space, allowing for the screening of many samples atone time.

The present invention provides a method for screening and selecting asoybean plant comprising QTL for disease resistance.

SUMMARY OF THE INVENTION

The present invention provides a method for assaying soybean plants fordisease resistance, immunity, or susceptibility comprising: (a)detaching a plant tissue from the soybean plant; (b) cultivating saidtissue in a media; (c) exposing said tissue to a plant pathogen; and (d)assessing said tissue for resistance, immunity, or susceptibility todisease caused by the pathogen. Additionally, the plant response to thepathogen can be evaluated by the following steps (e) isolating nucleicacids (DNA and/or RNA) from said plant; (f) assaying said nucleic acids(DNA, RNA, and/or cDNA) for the presence of one or more molecularmarkers for a quantitative trait locus associated with said resistance,immunity, or susceptibility; and (g) selecting said plant for use in abreeding program. Determination of resistance, immunity, orsusceptibility of a plant to a particular pathogen is obvious to anyoneskilled in the art. The plant tissue can be leaf, vascular tissue,flower, pod, root, stem, seed, or a portion thereof, or a cell isolatedfrom the tissue. Exposing said tissue to a plant pathogen isaccomplished by a means selected from the group consisting of (a) directapplication of the pathogen to the tissue; (b) inclusion of the pathogenin the culture media; and (c) inclusion of an agent that is effectivelycontaminated with the pathogen and serves to inoculate the tissue. Theplant pathogen can be a fungus, virus, bacterium, or invertebrateanimal. The plant pathogen exposure can be in the form of pathogenmacromolecules, cells, tissues, whole organism or combinations thereof,wherein the pathogen, and parts thereof, is either living or dead solong that the material mediates an immune response in the host tissue.Pathogen macromolecules relevant for the present invention include, butare not limited to, toxins, cell walls or membranes, antigens, andpolysaccharides.

The present invention further comprises a QTL that confers diseaseresistance to a fungal pathogen selected from the group consisting ofPhakopsora pachyrhizi, Phakopsora meibomiae (Asian Soybean Rust),Colletotrichum truncatum, Colletotrichum dematium var. truncatum,Glomerella glycines (Soybean Anthracnose), Phytophthora sojae(Phytophthora root and stem rot), Sclerotinia sclerotiorum (Sclerotiniastem rot), Fusarium solani f sp. glycines (sudden death syndrome),Fusarium spp. (Fusarium root rot), Macrophomina phaseolina (charcoalrot), Septoria glycines, (Brown Spot), Pythium aphanidermatum, Pythiumdebaryanum, Pythium irregulare, Pythium ultimum, Pythium myriotylum,Pythium torulosum (Pythium seed decay), Diaporthe phaseolorum var. sojae(Pod blight), Phomopsis longicola (Stem blight), Phomopsis spp.(Phomopsis seed decay), Peronospora manshurica (Downy Mildew),Rhizoctonia solani (Rhizoctonia root and stem rot, Rhizoctonia aerialblight), Phialophora gregata (Brown Stem Rot), Diaporthe phaseolorumvar. caulivora (Stem Canker), Cercospora kikuchii (Purple Seed Stain),Alternaria sp. (Target Spot), Cercospora sojina (Frogeye Leafspot),Sclerotium rolfsii (Southern blight), Arkoola nigra (Black leaf blight),Thielaviopsis basicola, (Black root rot), Choanephora infundibulifera,Choanephora trispora (Choanephora leaf blight), Leptosphaerulinatrifolii (Leptosphaerulina leaf spot), Mycoleptodiscus terrestris(Mycoleptodiscus root rot), Neocosmospora vasinfecta (Neocosmospora stemrot), Phyllosticta sojicola (Phyllosticta leaf spot), Pyrenochaetaglycines (Pyrenochaeta leaf spot), Cylindrocladium crotalariae (Redcrown rot), Dactuliochaeta glycines (Red leaf blotch), Spacelomaglycines (Scab), Stemphylium botryosum (Stemphylium leaf blight),Corynespora cassiicola (Target spot), Nematospora coryli (Yeast spot),and Phymatotrichum omnivorum (Cotton Root Rot).

The present invention further comprises a QTL that confers diseaseresistance to a viral pathogen selected from the group consisting ofAlfamovirus (Alfafa mosaic virus, AMV), Comovirus (bean pod mottlevirus, BPMV), Potyvirus (bean yellow mosaic virus, BYMV), Bromovirus(cowpea chlorotic mottle virus, CCMV), Begomovirus (mung bean yellowmosaivc virus, MYMV), Potyvirus (peanut mottle virus, PeMoV), Potyvirus(peanut stripe virus, PStV), Cucumovirus (peanut stunt virus, PSV),Caulimovirus (soybean chlorotic mottle virus, SbCMV), Begomovirus(soybean crinkle leaf virus, SCLV), Luteovirus (soybean dwarf virus,SbDV), Potyvirus (soybean mosaic virus, SMV), Nepovirus (soybean severestunt virus, SSSV), and Nepovirus (tobacco ringspot virus, TRSV).

The present invention further comprises a QTL that confers diseaseresistance to a bacterial pathogen selected from the group consisting ofBacillus subtilis (Bacillus seed decay), Pseudomonas savastonoi pv.glycinea (Bacterial blight), Pseudomonas syringae subsp. syringae(Bacterial crinkle-leaf), Xanthomonas axonopodis pv. glycines,(Bacterial pustule), Curtobacterium flaccumfaciens pv. flaccumfaciens,(Bacterial tan spot), Curtobacterium flaccumfaciens pv. flaccumfaciens,Ralstonia solanacearum, (Bacterial wilt), and Pseudomonas syringae pv.tabaci (Wildfire).

The present invention further comprises a QTL that confers diseaseresistance to a invertebrate pathogen selected from the group consistingof Aphis glycines (Soybean aphid), Heterodera glycines (Soybean cystnematode), Meloidogyne arenaria, Meloidogyne hapla, Meloidogyneincognita, Meloidogyne javanica (Root knot nematode), HoplolaimusColumbus, Hoplolaimus galeatus, Hoplolaimus magnistylus (Lancenematode), Pratylenchus spp. (Lesion nematode), Paratylenchus projectus,Paratylenchus tenuicaudatus (Pin nematode), Rotylenchulus reniformis(Reniform nematode), Criconemella ornata (Ring nematode),Hemicycliophora spp. (Sheath nematode), Heliocotylenchus spp. (Spiralnematode), Belonolainus gracilis, Belonolainus longicaudatus (Stingnematode), Quinisulcius acutus, Tylenchorhynchus spp. (Stunt nematode),and Paratrichodorus minor (Stubby root nematode).

The present invention also provides selected soybean tissue and plantsthat are resistant to Phakopsora pachyrhizi, Phakopsora meibomiae (AsianSoybean Rust), Colletotrichum truncatum, Colletotrichum dematium var.truncatum, Glomerella glycines (Soybean Anthracnose), Phytophthora sojae(Phytophthora root and stem rot), Sclerotinia sclerotiorum (Sclerotiniastem rot), Fusarium solani f. sp. glycines (sudden death syndrome),Fusarium spp. (Fusarium root rot), Macrophomina phaseolina (charcoalrot), Septoria glycines, (Brown Spot), Pythium aphanidermatum, Pythiumdebaryanum, Pythium irregulare, Pythium ultimum, Pythium myriotylum,Pythium torulosum (Pythium seed decay), Diaporthe phaseolorum var. sojae(Pod blight), Phomopsis longicola (Stem blight), Phomopsis spp.(Phomopsis seed decay), Peronospora manshurica (Downy Mildew),Rhizoctonia solani (Rhizoctonia root and stem rot, Rhizoctonia aerialblight), Phialophora gregata (Brown Stem Rot), Diaporthe phaseolorumvar. caulivora (Stem Canker), Cercospora kikuchii (Purple Seed Stain),Alternaria sp. (Target Spot), Cercospora sojina (Frogeye Leafspot),Sclerotium rolfsii (Southern blight), Arkoola nigra (Black leaf blight),Thielaviopsis basicola, (Black root rot), Choanephora infundibulifera,Choanephora trispora (Choanephora leaf blight), Leptosphaerulinatrifolii (Leptosphaerulina leaf spot), Mycoleptodiscus terrestris(Mycoleptodiscus root rot), Neocosmospora vasinfecta (Neocosmospora stemrot), Phyllosticta sojicola (Phyllosticta leaf spot), Pyrenochaetaglycines (Pyrenochaeta leaf spot), Cylindrocladium crotalariae (Redcrown rot), Dactuliochaeta glycines (Red leaf blotch), Spacelomaglycines (Scab), Stemphylium botryosum (Stemphylium leaf blight),Corynespora cassiicola (Target spot), Nematospora coryli (Yeast spot),Phymatotrichum omnivorum (Cotton Root Rot), Alfamovirus (Alfafa mosaicvirus, AMV), Comovirus (bean pod mottle virus, BPMV), Potyvirus (beanyellow mosaic virus, BYMV), Bromovirus (cowpea chlorotic mottle virus,CCMV), Begomovirus (mung bean yellow mosaivc virus, MYMV), Potyvirus(peanut mottle virus, PeMoV), Potyvirus (peanut stripe virus, PStV),Cucumovirus (peanut stunt virus, PSV), Caulimovirus (soybean chloroticmottle virus, SbCMV), Begomovirus (soybean crinkle leaf virus, SCLV),Luteovirus (soybean dwarf virus, SbDV), Potyvirus (soybean mosaic virus,SMV), Nepovirus (soybean severe stunt virus, SSSV), Nepovirus (tobaccoringspot virus, TRSV), Bacillus subtilis (Bacillus seed decay),Pseudomonas savastonoi pv. glycinea (Bacterial blight), Pseudomonassyringae subsp. syringae (Bacterial crinkle-leaf), Xanthomonasaxonopodis pv. glycines, (Bacterial pustule), Curtobacteriumflaccumfaciens pv. flaccumfaciens, (Bacterial tan spot), Curtobacteriumflaccumfaciens pv. flaccumfaciens, Ralstonia solanacearum, (Bacterialwilt), Pseudomonas syringae pv. tabaci (Wildfire), Aphis glycines(Soybean aphid), Heterodera glycines (Soybean cyst nematode),Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne incognita,Meloidogyne javanica (Root knot nematode), Hoplolaimus Columbus,Hoplolaimus galeatus, Hoplolaimus magnistylus (Lance nematode),Pratylenchus spp. (Lesion nematode), Paratylenchus projectus,Paratylenchus tenuicaudatus (Pin nematode), Rotylenchulus reniformis(Reniform nematode), Criconemella ornata (Ring nematode),Hemicycliophora spp. (Sheath nematode), Heliocotylenchus spp. (Spiralnematode), Belonolainus gracilis, Belonolainus longicaudatus (Stingnematode), Quinisulcius acutus, Tylenchorhynchus spp. (Stunt nematode),or Paratrichodorus minor (Stubby root nematode).

The present invention further provides that the selected plant is fromthe group consisting of members of the genus Glycine, more specificallyfrom the group consisting of Glycine arenaria, Glycine argyrea, Glycinecanescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba,Glycine falcate, Glycine latifolia, Glycine latrobeana, Glycine max,Glycine microphylla, Glycine pescadrensis, Glycine pindanica, Glycinerubiginosa, Glycine soja, Glycine sp., Glycine stenophita, Glycinetabacina and Glycine tomentella.

The present invention further provides that the media used in the methodfor selection is comprised of water that is untreated, distilled ordeionized. The media can contain any ingredients necessary to sustainthe pathogen or plant tissue, so long as the ingredients do notinterfere with the expression of resistance as conferred by the QTL.

The present invention further provides a soybean plant selected usingsaid method.

The present invention also provides a QTL that is selected from thegroup consisting of Phytophthora (root rot) infection tolerance locus,Fusarium solani f sp. glycines (sudden death syndrome) resistance locus,Cercospora sojina (Frogeye leaf spot) resistance locus, Phialophoragegata (brown stem rot) resistance locus, Sclerotinia (stem rot)resistance locus, ASR resistance locus 1, ASR resistance locus 2, ASRresistance locus 3, ASR resistance locus 4, ASR resistance locus 5, ASRresistance locus 6, ASR resistance locus 7, ASR resistance locus 8, ASRresistance locus 9, ASR resistance locus 10, ASR resistance locus 11,ASR resistance locus 12, and ASR resistance locus 13.

The present invention further provides that the selected plant containsone or more fungal disease resistance QTL, including ASR resistancelocus 1, ASR resistance locus 2, ASR resistance locus 3, ASR resistancelocus 4, ASR resistance locus 5, ASR resistance locus 6, ASR resistancelocus 7, ASR resistance locus 8, ASR resistance locus 9, ASR resistancelocus 10, ASR resistance locus 11, ASR resistance locus 12, and ASRresistance locus 13.

The present invention further provides one or more single nucleotidepolymorphism (SNP) marker loci for ASR resistance locus 1, wherein saidSNP marker is selected from the group consisting of NS0093250,NS0119710, NS0103004, NS0099454, NS0102630, NS0102915, NS0102913,NS0123728, NS0129943, NS0102168, NS0092723, NS0098177, NS0127343, andNS0101121. One or more SNP marker loci for ASR resistance locus 3 arealso provided, wherein said SNP marker is selected from the groupconsisting of NS0099746, NS0123747, NS0126598, NS0128378, NS0096829,NS0125408, NS0098902, NS0099529, NS0097798, NS0137477, NS0095322,NS0136101, and NS0098982. An exemplary SNP marker locus, NS0103033, isprovided for ASR resistance locus 5, ASR resistance locus 6, ASRresistance locus 7, ASR resistance locus 8, and ASR resistance locus 9.Another exemplary SNP marker locus, NS0124935, is provided for ASRresistance locus 10, ASR resistance locus 11, ASR resistance locus 12,and ASR resistance locus 13. Further, one or more markers mapped within10 centimorgans or less from said marker molecules can be used for theselection and introgression of ASR resistance loci.

The present invention further provides a method for selecting andintrogressing ASR resistance in soybean comprising: (a) isolatingnucleic acids from a plurality of soybean plants; (b) detecting in saidisolated nucleic acids the presence of one or more marker moleculesassociated with ASR resistance loci 1-13, wherein said marker moleculeis selected from the group consisting of SEQ ID NOs: 67 through 99, andany one marker molecule mapped within 10 centimorgans or less from saidmarker molecules; and (c) selecting a soybean plant comprising said oneor more marker molecules, thereby selecting an ASR resistant soybeanplant.

The present invention further provides for a soybean plant selectedusing said method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for screening soybean plants forresistance, immunity, or susceptibility to a fungal disease. In apreferred embodiment the plant is selected from the genus Glycine. Thewild perennial soybeans belong to the subgenus Glycine and have a widearray of genetic diversity. The cultivated soybean (Glycine max (L.)Merr.) and its wild annual progenitor (Glycine soja (Sieb. and Zucc.))belong to the subgenus Soja, contain 2n=40 chromosomes, arecross-compatible, usually produce vigorous fertile F1 hybrids, and carrysimilar genomes. Crosses between cultivated Glycine species and wildperennial Glycine species are possible, the success of which is variableamongst accessions. Investigations have shown that several wildperennial Glycine accessions carry resistance to brown spot, soybeanrust, root rot, yellow mosaic virus, and powdery mildew. There are morethan 100,000 Glycine max accessions, probably less than 10,000 Glycinesoja accessions and approximately, 3500 accessions of perennial Glycinespecies in germplasm collections throughout the world. The exact numbersare unknown. Major Glycine collections exist in Australia, Brazil,China, Germany, India, Indonesia, Japan, Russia, South Korea, and theUnited States. Many other smaller but important collections existthroughout Asia and Europe. It is not known how many of the accessionsare duplicated among collections. The USDA Soybean Germplasm Collectionis one of the largest collections and the largest outside Asia (Verma etal., eds., Soybean: Genetics, Molecular Biology and Biotechnology(1996)). It currently contains 20,765 accessions, comprised of 19species collections, including 18,680 accessions of Glycine max and1,166 accessions of Glycine soja as well as perennial Glycine species.

In a preferred embodiment, a soybean plant is assayed for diseaseresistance, immunity, or susceptibility comprising: (a) detaching aplant tissue from the soybean plant; (b) cultivating said tissue in amedia; (c) exposing said tissue to a plant pathogen; and (d) assessingsaid tissue for resistance, immunity, or susceptibility to diseasecaused by the pathogen. Additionally, the plant response to the pathogencan be evaluated by the following steps (e) isolating nucleic acids fromsaid plant; (f) assaying said nucleic acids for the presence of one ormore molecular markers for a quantitative trait locus associated withsaid resistance, immunity, or susceptibility; and (g) selecting saidplant for use in a breeding program. Determination of resistance,immunity, or susceptibility of a plant to a particular pathogen isobvious to anyone skilled in the art. The plant tissue can be leaf,vascular tissue, flower, pod, root, stem, seed, or a portion thereof, ora cell isolated from the tissue. Exposing said tissue to a plantpathogen is accomplished by a means selected from the group consistingof (a) direct application of the pathogen to the tissue; (b) inclusionof the pathogen in the culture media; and (c) inclusion of an agent thatis effectively contaminated with the pathogen and serves to inoculatethe tissue. The plant pathogen can be a fungus, virus, bacterium, orinvertebrate animal. The plant pathogen exposure can be in the form ofpathogen macromolecules, cells, tissues, whole organism or combinationsthereof, wherein the pathogen, and parts thereof, is either living ordead so long that the material mediates an immune response in the hosttissue. Pathogen macromolecules relevant for the present inventioninclude, but are not limited to, toxins, cell walls or membranes,antigens, and polysaccharides.

In a preferred embodiment, the leaf tissue may comprise a cotyledonleaf, unifoliate leaf, a trifoliate leaf, and prophylls. There are fourtypes of soybean leaves: 1) the first pair of simple cotyledons or seedleaves, 2) second pair of simple primary leaves, also known asunifoliate leaves, 3) trifoliate foliage leaves, and 4) prophylls, whichare plant parts resembling leaves. The unifoliate leaves occur at thefirst node above the cotyledons. All other leaves would be trifoliates,wherein the first pair to emerge following the unifoliates are the firsttrifoliate leaves, which are followed by the emergence of the secondtrifoliates leaves and then the third trifoliate leaves (H. R. Boermaand J. E. Specht (ed.) Soybean Monograph, 3rd Edition, Am. Soc. Agron.,Madison, Wis. (2004)).

In a preferred embodiment, the present invention enables a soybean plantto be assayed for resistance, immunity, or susceptibility to a fungaldisease. Soybean diseases caused by fungi include, but are not limitedto, Phakopsora pachyrhizi, Phakopsora meibomiae (Asian Soybean Rust),Colletotrichum truncatum, Colletotrichum dematium var. truncatum,Glomerella glycines (Soybean Anthracnose), Phytophthora sojae(Phytophthora root and stem rot), Sclerotinia sclerotiorum (Sclerotiniastem rot), Fusarium solani f sp. glycines (sudden death syndrome),Fusarium spp. (Fusarium root rot), Macrophomina phaseolina (charcoalrot), Septoria glycines, (Brown Spot), Pythium aphanidermatum, Pythiumdebaryanum, Pythium irregulare, Pythium ultimum, Pythium myriotylum,Pythium torulosum (Pythium seed decay), Diaporthe phaseolorum var. sojae(Pod blight), Phomopsis longicola (Stem blight), Phomopsis spp.(Phomopsis seed decay), Peronospora manshurica (Downy Mildew),Rhizoctonia solani (Rhizoctonia root and stem rot, Rhizoctonia aerialblight), Phialophora gregata (Brown Stem Rot), Diaporthe phaseolorumvar. caulivora (Stem Canker), Cercospora kikuchii (Purple Seed Stain),Alternaria sp. (Target Spot), Cercospora sojina (Frogeye Leafspot),Sclerotium rolfsii (Southern blight), Arkoola nigra (Black leaf blight),Thielaviopsis basicola, (Black root rot), Choanephora infundibulifera,Choanephora trispora (Choanephora leaf blight), Leptosphaerulinatrifolii (Leptosphaerulina leaf spot), Mycoleptodiscus terrestris(Mycoleptodiscus root rot), Neocosmospora vasinfecta (Neocosmospora stemrot), Phyllosticta sojicola (Phyllosticta leaf spot), Pyrenochaetaglycines (Pyrenochaeta leaf spot), Cylindrocladium crotalariae (Redcrown rot), Dactuliochaeta glycines (Red leaf blotch), Spacelomaglycines (Scab), Stemphylium botryosum (Stemphylium leaf blight),Corynespora cassiicola (Target spot), Nematospora coryli (Yeast spot),and Phymatotrichum omnivorum (Cotton Root Rot).

In a preferred embodiment, the present invention enables a soybean plantto be assayed for resistance, immunity, or susceptibility to a viraldisease. Soybean diseases caused by viruses include, but are not limitedto, Alfamovirus (Alfafa mosaic virus, AMV), Comovirus (bean pod mottlevirus, BPMV), Potyvirus (bean yellow mosaic virus, BYMV), Bromovirus(cowpea chlorotic mottle virus, CCMV), Begomovirus (mung bean yellowmosaivc virus, MYMV), Potyvirus (peanut mottle virus, PeMoV), Potyvirus(peanut stripe virus, PStV), Cucumovirus (peanut stunt virus, PSV),Caulimovirus (soybean chlorotic mottle virus, SbCMV), Begomovirus(soybean crinkle leaf virus, SCLV), Luteovirus (soybean dwarf virus,SbDV), Potyvirus (soybean mosaic virus, SMV), Nepovirus (soybean severestunt virus, SSSV), and Nepovirus (tobacco ringspot virus, TRSV).

In a preferred embodiment, the present invention enables a soybean plantto be assayed for resistance, immunity, or susceptibility to a bacterialdisease. Soybean diseases caused by bacteria include, but are notlimited to, Bacillus subtilis (Bacillus seed decay), Pseudomonassavastonoi pv. glycinea (Bacterial blight), Pseudomonas syringae subsp.syringae (Bacterial crinkle-leaf), Xanthomonas axonopodis pv. glycines,(Bacterial pustule), Curtobacterium flaccumfaciens pv. flaccumfaciens,(Bacterial tan spot), Curtobacterium flaccumfaciens pv. flaccumfaciens,Ralstonia solanacearum, (Bacterial wilt), and Pseudomonas syringae pv.tabaci (Wildfire).

In a preferred embodiment, the present invention enables a soybean plantto be assayed for resistance, immunity, or susceptibility to an animalpest disease. Soybean diseases caused by animal pests include, but arenot limited to Aphis glycines (Soybean aphid), Heterodera glycines(Soybean cyst nematode), Meloidogyne arenaria, Meloidogyne hapla,Meloidogyne incognita, Meloidogyne javanica (Root knot nematode),Hoplolaimus Columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus(Lance nematode), Pratylenchus spp. (Lesion nematode), Paratylenchusprojectus, Paratylenchus tenuicaudatus (Pin nematode), Rotylenchulusreniformis (Reniform nematode), Criconemella ornata (Ring nematode),Hemicycliophora spp. (Sheath nematode), Heliocotylenchus spp. (Spiralnematode), Belonolainus gracilis, Belonolainus longicaudatus (Stingnematode), Quinisulcius acutus, Tylenchorhynchus spp. (Stunt nematode),and Paratrichodorus minor (Stubby root nematode).

The invention further provides a method for selection and introgressionof QTL for disease resistance in soybean comprising: (a) isolatingnucleic acids from a plurality of soybean plants; (b) detecting in saidisolated nucleic acids the presence of one or more marker moleculesassociated with disease resistance QTL; and (c) selecting a soybeanplant comprising said one or more marker molecules, thereby selecting adisease resistant soybean plant.

The disease resistance QTL of the present invention may be introducedinto an elite Glycine max line. An “elite line” is any line that hasresulted from breeding and selection for superior agronomic performance.Examples of elite lines are lines that are commercially available tofarmers or soybean breeders such as HARTZ™ variety H4994, HARTZ™ varietyH5218, HARTZ™ variety H5350, HARTZ™ variety H5545, HARTZ™ variety H5050,HARTZ™ variety H5454, HARTZ™ variety H5233, HARTZ™ variety H5488, HARTZ™variety HLA572, HARTZ™ variety H6200, HARTZ™ variety H6104, HARTZ™variety H6255, HARTZ™ variety H6586, HARTZ™ variety H6191, HARTZ™variety H7440, HARTZ™ variety H4452 ROUNDUP READY™, HARTZ™ variety H4994ROUNDUP READY™, HARTZ™ variety H4988 ROUNDUP READY™, HARTZ™ varietyH5000 ROUNDUP READY™, HARTZ™ variety H5147 ROUNDUP READY™ HARTZ™ varietyH5247 ROUNDUP READY™, HARTZ™ variety H5350 ROUNDUP READY™, HARTZ™variety H5545 ROUNDUP READY™, HARTZ™ variety H5855 ROUNDUP READY™,HARTZ™ variety H5088 ROUNDUP READY™, HARTZ™ variety H5164 ROUNDUPREADY™, HARTZ™ variety H5361 ROUNDUP READY™, HARTZ™ variety H5566ROUNDUP READY™ HARTZ™ variety H5181 ROUNDUP READY™, HARTZ™ variety H5889ROUNDUP READY™, HARTZ™ variety H5999 ROUNDUP READY™, HARTZ™ varietyH6013 ROUNDUP READY™, HARTZ™ variety H6255 ROUNDUP READY™, HARTZ™variety H6454 ROUNDUP READY™, HARTZ™ variety H6686 ROUNDUP READY™,HARTZ™ variety H7152 ROUNDUP READY™ HARTZ™ variety H7550 ROUNDUP READY™,HARTZ™ variety H8001 ROUNDUP READY™ (HARTZ SEED, Stuttgart, Ark., USA);A0868, AG0202, AG0401, AG0803, AG0901, A1553, A1900, AG1502, AG1702,AG1901, A1923, A2069, AG2101, AG2201, AG2205, A2247, AG2301, A2304,A2396, AG2401, AG2501, A2506, A2553, AG2701, AG2702, AG2703, A2704,A2833, A2869, AG2901, AG2902, AG2905, AG3001, AG3002, AG3101, A3204,A3237, A3244, AG3301, AG3302, AG3006, AG3203, A3404, A3469, AG3502,AG3503, AG3505, AG3305, AG3602, AG3802, AG3905, AG3906, AG4102, AG4201,AG4403, AG4502, AG4603, AG4801, AG4902, AG4903, AG5301, AG5501, AG5605,AG5903, AG5905, A3559, AG3601, AG3701, AG3704, AG3750, A3834, AG3901,A3904, A4045 AG4301, A4341, AG4401, AG4404, AG4501, AG4503, AG4601,AG4602, A4604, AG4702, AG4703, AG4901, A4922, AG5401, A5547, AG5602,AG5702, A5704, AG5801, AG5901, A5944, A5959, AG6101, AJW2600COR,FPG26932, QR4459 and QP4544 (Asgrow Seeds, Des Moines, Iowa, USA);DKB26-52, DKB28-51, DKB32-52, DKB08-51, DKB09-53, DKB10-52, DKB18-51,DKB26-53, DKB29-51, DKB42-51, DKB35-51 DKB34-51, DKB36-52, DKB37-51,DKB38-52, DKB46-51, DKB54-52 and DeKalb variety CX445 (DeKalb, Ill.,USA); 91B91, 92B24, 92B37, 92B63, 92B71, 92B74, 92B75, 92B91, 93B01,93B11, 93B26, 93B34, 93B35, 93B41, 93B45, 93B51, 93B53, 93B66, 93B81,93B82, 93B84, 94B01, 94B32, 94B53, 94M80 RR, 94M50 RR, 95B71, 95B95,95M81 RR, 95M50 RR, 95M30 RR, 9306, 9294, 93M50, 93M93, 94B73, 94B74,94M41, 94M70, 94M90, 95B32, 95B42, 95B43 and 9344 (Pioneer Hi-bredInternational, Johnston, Iowa, USA); SSC-251RR, SSC-273CNRR, AGRA5429RR, SSC-314RR, SSC-315RR, SSC-311STS, SSC-320RR, AGRA5432RR,SSC-345RR, SSC-356RR, SSC-366, SSC-373RR and AGRA5537CNRR (SchlessmanSeed Company, Milan, Ohio, USA); 39-E9, 44-R4, 44-R5, 47-G7, 49-P9,52-Q2, 53-K3, 56-J6, 58-V8, ARX A48104, ARX B48104, ARX B55104 and GP530(Armor Beans, Fisher, Ark., USA); HT322STS, HT3596STS, L0332, L0717,L1309CN, L1817, L1913CN, L1984, L2303CN, L2495, L2509CN, L2719CN,L3997CN, L4317CN, RC1303, RC1620, RC1799, RC1802, RC1900, RC1919,RC2020, RC2300, RC2389, RC2424, RC2462, RC2500, RC2504, RC2525, RC2702,RC2964, RC3212, RC3335, RC3354, RC3422, RC3624, RC3636, RC3732, RC3838,RC3864, RC3939, RC3942, RC3964, RC4013, RC4104, RC4233, RC4432, RC4444,RC4464, RC4842, RC4848, RC4992, RC5003, RC5222, RC5332, RC5454, RC5555,RC5892, RC5972, RC6767, RC7402, RT0032, RT0041, RT0065, RT0073, RT0079,RT0255, RT0269, RT0273, RT0312, RT0374, RT0396, RT0476, RT0574, RT0583,RT0662, RT0669, RT0676, RT0684, RT0755, RT0874, RT0907, RT0929, RT0994,RT0995, RT1004, RT1183, RT1199, RT1234, RT1399, RT1413, RT1535, RT1606,RT1741, RT1789, RT1992, RT2000, RT2041, RT2089, RT2092, RT2112, RT2127,RT2200, RT2292, RT2341, RT2430, RT2440, RT2512, RT2544, RT2629, RT2678,RT2732, RT2800, RT2802, RT2822, RT2898, RT2963, RT3176, RT3200, RT3253,RT3432, RT3595, RT3836, RT4098, RX2540, RX2944, RX3444 and TS466RR(Croplan Genetics, Clinton, Ky., USA); 4340RR, 4630RR, 4840RR, 4860RR,4960RR, 4970RR, 5260RR, 5460RR, 5555RR, 5630RR and 5702RR (Delta Grow,England, Ark., USA); DK3964RR, DK3968RR, DK4461RR, DK4763RR, DK4868RR,DK4967RR, DK5161RR, DK5366RR, DK5465RR, DK55T6, DK5668RR, DK5767RR,DK5967RR, DKXTJ446, DKXTJ448, DKXTJ541, DKXTJ542, DKXTJ543, DKXTJ546,DKXTJ548, DKXTJ549, DKXTJ54J9, DKXTJ54X9, DKXTJ554, DKXTJ555, DKXTJ55J5and DKXTJ5K57 (Delta King Seed Company, McCrory, Ark., USA); DP 3861RR,DP 4331 RR, DP 4546RR, DP 4724 RR, DP 4933 RR, DP 5414RR, DP 5634 RR, DP5915 RR, DPX 3950RR, DPX 4891RR, DPX 5808RR (Delta & Pine Land Company,Lubbock, Tex., USA); DG31T31, DG32C38, DG3362NRR, DG3390NRR, DG33A37,DG33B52, DG3443NRR, DG3463NRR, DG3481NRR, DG3484NRR, DG3535NRR,DG3562NRR, DG3583NRR, DG35B40, DG35D33, DG36M49, DG37N43, DG38K57,DG38T47, SX04334, SX04453 (Dyna-n) gro line, UAP-MidSouth, Cordova,Tenn., USA); 8374RR CYSTX, 8390 NNRR, 8416RR, 8492NRR and 8499NRR (ExcelBrand, Camp Point, Ill., USA); 4922RR, 5033RR, 5225RR and 5663RR (FFRSeed, Southhaven, Miss., USA); 3624RR/N, 3824RR/N, 4212RR/N, 4612RR/N,5012RR/N, 5212RR/N and 5412RR/STS/N (Garst Seed Company, Slater, Iowa,USA); 471, 4R451, 4R485, 4R495, 4RS421 and 5R531 (Gateway Seed Company,Nashville, Ill., USA); H-3606RR, H-3945RR, H-4368RR, H-4749RR, H-5053RRand H-5492RR (Golden Harvest Seeds, Inc., Pekin, Ill., USA); HBK 5324,HBK 5524, HBK R4023, HBK R4623, HBK R4724, HBK R4820, HBK R4924, HBKR4945CX, HBK R5620 and HBK R5624 (Hornbeck Seed Co. Inc., DeWitt, Ark.,USA); 341 RR/SCN, 343 RR/SCN, 346 RR/SCN, 349 RR, 355 RR/SCN, 363RR/SCN, 373 RR, 375 RR, 379 RR/SCN, 379+ RR/SCN, 380 RR/SCN, 380+RR/SCN, 381 RR/SCN, 389 RR/SCN, 389+ RR/SCN, 393 RR/SCN, 393+ RR/SCN,398 RR, 402 RR/SCN, 404 RR, 424 RR, 434 RR/SCN and 442 RR/SCN (KrugerSeed Company, Dike, Iowa, USA); 3566, 3715, 3875, 3944, 4010 and 4106(Lewis Hybrids, Inc., Ursa, Ill., USA); C3999NRR (LG Seeds, Elmwood,Ill., USA); Atlanta 543, Austin RR, Cleveland VIIRR, Dallas RR, DenverRRSTS, Everest RR, Grant 3RR, Olympus RR, Phoenix IIIRR, Rocky RR,Rushmore 553RR and Washington IXRR (Merschman Seed Inc., West Point,Iowa, USA); RT 3304N, RT 3603N, RT 3644N, RT 3712N, RT 3804N, RT 3883N,RT 3991N, RT 4044N, RT 4114N, RT 4124N, RT 4201N, RT 4334N, RT 4402N, RT4480N, RT 4503N, RT 4683N, RT 4993N, RT 5043N, RT 5204, RT 5553N, RT5773, RT4731N and RTS 4824N (MFA Inc., Columbia, Mo., USA); 9A373NRR,9A375XRR, 9A385NRS, 9A402NRR, 9A455NRR, 9A485XRR and 9B445NRS (MidlandGenetics Group L.L.C., Ottawa, Kans., USA); 3605nRR, 3805nRR, 3903nRR,3905nRR, 4305nRR, 4404nRR, 4705nRR, 4805nRR, 4904nRR, 4905nRR, 5504nRRand 5505nRR (Midwest Premium Genetics, Concordia, Mo., USA); S37-N4,S39-K6, S40-R9, S42-P7, S43-B1, S49-Q9, S50-N3, S52-U3 and S56-D7(Syngenta Seeds, Henderson, Ky., USA); NT-3707 RR, NT-3737 RR/SCN,NT-3737+ RR/SCN, NT-3737sc RR/SCN, NT-3777+ RR, NT-3787 RR/SCN, NT-3828RR, NT-3839 RR, NT-3909 RR/SCN/STS, NT-3909+ RR/SCN/ST, NT-3909scRR/SCN/S, NT-3919 RR, NT-3922 RR/SCN, NT-3929 RR/SCN, NT-3999 RR/SCN,NT-3999+ RR/SCN, NT-3999sc RR/SCN, NT-4040 RR/SCN, NT-4040+ RR/SCN,NT-4044 RR/SCN, NT-4122 RR/SCN, NT-4414 RR/SCN/STS, NT-4646 RR/SCN andNT-4747 RR/SCN (NuTech Seed Co., Ames, Iowa, USA); PB-3494NRR,PB-3732RR, PB-3894NRR, PB-3921NRR, PB-4023NRR, PB-4394NRR, PB-4483NRRand PB-5083NRR (Prairie Brand Seed Co., Story City, Iowa, USA); 3900RR,4401RR, 4703RR, 4860RR, 4910, 4949RR, 5250RR, 5404RR, 5503RR, 5660RR,5703RR, 5770, 5822RR, PGY 4304RR, PGY 4604RR, PGY 4804RR, PGY 5622RR andPGY 5714RR (Progeny Ag Products, Wynne, Ark., USA); R3595RCX, R3684Rcn,R3814RR, R4095Rcn, R4385Rcn and R4695Rcn (Renze Hybrids Inc., Carroll,Iowa, USA); S3532-4, S3600-4, S3832-4, S3932-4, S3942-4, S4102-4,S4542-4 and S4842-4 (Stine Seed Co., Adel, Iowa, USA); 374RR, 398RRS(Taylor Seed Farms Inc., White Cloud, Kans., USA); USG 5002T, USG510nRR, USG 5601T, USG 7440nRR, USG 7443nRR, USG 7473nRR, USG 7482nRR,USG 7484nRR, USG 7499nRR, USG 7504nRR, USG 7514nRR, USG 7523nRR, USG7553nRS and USG 7563nRR (UniSouth Genetics Inc., Nashville, Tenn., USA);V38N5RS, V39N4RR, V42N3RR, V48N5RR, V284RR, V28N5RR, V315RR, V35N4RR,V36N5RR, V37N3RR, V40N3RR, V47N3RR, and V562NRR (Royster-Clark Inc.,Washington C. H., Ohio, USA); RR2383N, 2525NA, RR2335N, RR2354N,RR2355N, RR2362, RR2385N, RR2392N, RR2392NA, RR2393N, RR2432N, RR2432NA,RR2445N, RR2474N, RR2484N, RR2495N and RR2525N (Willcross Seed, KingCity Seed, King City, Mo., USA); 1493RR, 1991NRR, 2217RR, 2301NRR,2319RR, 2321NRR, 2341NRR, 2531NRR, 2541NRR, 2574RR, 2659RR, 2663RR,2665NRR, 2671NRR, 2678RR, 2685RR, 2765NRR, 2782NRR, 2788NRR, 2791NRR,3410RR, 3411NRR, 3419NRR, 3421NRR, 3425NRR, 3453NRR, 3461NRR, 3470CRR,3471NRR, 3473NRR, 3475RR, 3479NRR, 3491NRR, 3499NRR, WX134, WX137, WX177and WX300 (Wilken Seeds, Pontiac, Ill., USA). An elite plant is arepresentative plant from an elite line.

The disease resistance QTL of the present invention may also beintroduced into an elite Glycine max transgenic plant that contains oneor more genes for herbicide tolerance, increased yield, insect control,fungal disease resistance, virus resistance, nematode resistance,bacterial disease resistance, mycoplasma disease resistance, modifiedoils production, high oil production, high protein production,germination and seedling growth control, enhanced animal and humannutrition, low raffinose, environmental stress resistant, increaseddigestibility, industrial enzymes, pharmaceutical proteins, peptides andsmall molecules, improved processing traits, improved flavor, nitrogenfixation, hybrid seed production, reduced allergenicity, biopolymers,and biofuels among others. These agronomic traits can be provided by themethods of plant biotechnology as transgenes in Glycine max.

It is further understood that a soybean plant of the present inventionmay exhibit the characteristics of any maturity group. The pollen fromthe selected soybean plant can be cryopreserved and used in crosses withelite lines from other maturity groups to introgress a the fungaldisease resistance locus into a line that would not normally beavailable for crossing in nature. Pollen cryopreservation techniques arewell known in the art (Tyagi et al., Cryo Letters, 24: 119-124 (2003),Liang et al., Acta Botanica Sinica, 35: 733-738 (1993), and Honda etal., Euphytica 126: 315-320 (2002)).

The disease resistant effect of the QTL can vary based on the parentalgenotype and on the environmental conditions in which the diseaseresistance effect is measured. It is within the skill of those in theart of plant breeding and without undue experimentation to use themethods described herein to select from a population of plants or from acollection of parental genotypes those that when containing a diseaselocus result in enhanced disease resistance relative to the parentgenotype. Herein, a plant disease can be caused by a fungi, virus,bacterium or invertebrate animal.

A number of molecular genetic maps of Glycine have been reported (Mansuret al., Crop Sci. 36: 1327-1336 (1996), Shoemaker et al., Genetics 144:329-338 (1996), Shoemaker et al., Crop Science 32: 1091-1098 (1992),Shoemaker et al., Crop Science 35: 436-446 (1995), Tinley et al., J.Cell Biochem. Suppl. 14E: 291 (1990), Cregan et al., Crop Science39:1464-1490 (1999)). Glycine max, Glycine soja and Glycine max x.Glycine soja share linkage groups (Shoemaker et al., Genetics 144:329-338 (1996)). As used herein, reference to the linkage groups, G; C2;J; and N of Glycine max refers to the linkage group that corresponds tolinkage groups, G; C2; J; and N from the genetic map of Glycine max(Mansur et al., Crop Science. 36: 1327-1336 (1996), Cregan et al., CropScience 39:1464-1490 (1999), and Soybase, Agricultural Research Service,United States Department of Agriculture).

An allele of a QTL can, of course, comprise multiple genes or othergenetic factors even within a contiguous genomic region or linkagegroup, such as a haplotype. As used herein, an allele of a QTL cantherefore encompasses more than one gene or other genetic factor whereeach individual gene or genetic component is also capable of exhibitingallelic variation and where each gene or genetic factor is also capableof eliciting a phenotypic effect on the quantitative trait in question.In an embodiment of the present invention the allele of a QTL comprisesone or more genes or other genetic factors that are also capable ofexhibiting allelic variation. The use of the term “an allele of a QTL”is thus not intended to exclude a QTL that comprises more than one geneor other genetic factor. Specifically, an “allele of a QTL” in thepresent in the invention can denote a haplotype within a haplotypewindow wherein a phenotype can be disease resistance. A haplotype windowis a contiguous genomic region that can be defined, and tracked, with aset of one or more polymorphic markers wherein said polymorphismsindicate identity by descent. A haplotype within that window can bedefined by the unique fingerprint of alleles at each marker. As usedherein, an allele is one of several alternative forms of a geneoccupying a given locus on a chromosome. When all the alleles present ata given locus on a chromosome are the same, that plant is homozygous atthat locus. If the alleles present at a given locus on a chromosomediffer, that plant is heterozygous at that locus.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). A cultivar is a race or variety of a plantthat has been created or selected intentionally and maintained throughcultivation.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) of the progeny of any cross. It isfurther understood that any commercial and non-commercial cultivars canbe utilized in a breeding program. Factors such as, for example,emergence vigor, vegetative vigor, stress tolerance, disease resistance,branching, flowering, seed set, seed size, seed density, standability,and threshability etc. will generally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredembodiment a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollinationevent, and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids are selected for certain single gene traitssuch as pod color, flower color, seed yield, pubescence color orherbicide resistance which indicate that the seed is truly a hybrid.Additional data on parental lines, as well as the phenotype of thehybrid, influence the breeder's decision whether to continue with thespecific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals in thebest families is selected. Replicated testing of families can begin inthe F₄ generation to improve the effectiveness of selection for traitswith low heritability. At an advanced stage of inbreeding (i.e., F₆ andF₇), the best lines or mixtures of phenotypically similar lines aretested for potential release as new cultivars.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seed of apopulation each generation of inbreeding.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Fehr, Principles of Cultivar Development Vol. 1, pp. 2-3(1987)).

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred embodiment ofthe present invention, the plant part is a seed.

Plants or parts thereof of the present invention may be grown in cultureand regenerated. Methods for the regeneration of Glycine max plants fromvarious tissue types and methods for the tissue culture of Glycine maxare known in the art (See, for example, Widholm et al., In VitroSelection and Culture-induced Variation in Soybean, In Soybean:Genetics, Molecular Biology and Biotechnology, eds. Verma and Shoemaker,CAB International, Wallingford, Oxon, England (1996)). Regenerationtechniques for plants such as Glycine max can use as the startingmaterial a variety of tissue or cell types. With Glycine max inparticular, regeneration processes have been developed that begin withcertain differentiated tissue types such as meristems (Cartha et al.,Can. J. Bot. 59:1671-1679 (1981)), hypocotyl sections (Cameya et al.,Plant Science Letters 21: 289-294 (1981)), and stem node segments (Sakaet al., Plant Science Letters, 19: 193-201 (1980), Cheng et al., PlantScience Letters, 19: 91-99 (1980)). Regeneration of whole sexuallymature Glycine max plants from somatic embryos generated from explantsof immature Glycine max embryos has been reported (Ranch et al., InVitro Cellular & Developmental Biology 21: 653-658 (1985)). Regenerationof mature Glycine max plants from tissue culture by organogenesis andembryogenesis has also been reported (Barwale et al., Planta 167:473-481 (1986), Wright et al., Plant Cell Reports 5: 150-154 (1986)).

The present invention also provides a disease resistant soybean plantselected for by screening for disease resistance, immunity, orsusceptibility in the soybean plant, the selection comprisinginterrogating genomic nucleic acids for the presence of a markermolecule that is genetically linked to an allele of a QTL associatedwith disease resistance in the soybean plant, where the allele of a QTLis also located on a linkage group associated with disease resistantsoybean. The disease can be caused by a fungus, virus, bacterium, orinvertebrate animal.

The present invention also provides for QTL conferring resistance toAsian Soybean Rust, including ASR resistance locus 1, ASR resistancelocus 2, ASR resistance locus 3, ASR resistance locus 4, ASR resistancelocus 5, ASR resistance locus 6, ASR resistance locus 7, ASR resistancelocus 8, ASR resistance locus 9, ASR resistance locus 10, ASR resistancelocus 11, ASR resistance locus 12, and ASR resistance locus 13. Fourdominant and independently inherited loci for resistance to P.pachyrhizi, herein designated ASR resistance locus 1 through 4, havebeen identified in PI 200492, PI 230970, PI 462312 (Ankur), and PI459025B, respectively. In the present invention, ASR resistance locus 1has been localized to linkage group G of soybean. SNP markers used tomonitor the introgression of ASR resistance locus 1 are selected fromthe group consisting of NS0093250, NS0119710, NS0103004, NS0099454,NS0102630, NS0102915, NS0102913, NS0123728, NS0129943, NS0102168,NS0092723, NS0098177, NS0127343 and NS0101121. The ASR resistance locus1 SNP marker DNA sequences (presented as SEQ ID NOs: 67 through 80) canbe amplified using the primers indicated as SEQ ID NOs: 1 through 28 anddetected with probes indicated as SEQ ID NOs: 100 through 127. In thepresent invention, ASR resistance locus 2 is most likely located onlinkage group J, near or within the disease resistance clustercontaining Brown Stem Rot, Soybean Cyst Nematode resistance and Frog EyeLeaf Spot resistance; or linkage group N. In the present invention, ASRresistance locus 3 is localized to linkage group C2. SNP markers used tomonitor the introgression of ASR resistance locus 3 are selected fromthe group consisting of NS0099746, NS0123747, NS0126598, NS0128378,NS0096829, NS0125408, NS0098902, NS0099529, NS0097798, NS0137477,NS0095322, NS0136101, NS0098982, NS0103749, NS0118897, NS0119715, andNS0130920. These marker DNA sequences (presented as SEQ ID NOs:81through 97) can be amplified using the primers indicated as SEQ ID NOs:29 through 62 and detected with probes indicated as SEQ ID NOs: 128through 161. In the present invention, ASR resistance locus 4 is likelylocated on linkage group N.

The present invention also provides for haplotypes that conferresistance to ASR that were identified in association studies. Thesegenome-wide surveys revealed two SNP markers associated with ASRresistance located in two different windows on chromosome 13. In thefirst haplotype window, the SNP marker used to monitor he introgressionof ASR resistance locus 5, ASR resistance locus 6, ASR resistance locus7, ASR resistance locus 8, and ASR resistance locus 9 is NS0103033. ThisSNP marker DNA sequences (presented as SEQ ID NO: 98) can be amplifiedusing the primers indicated as SEQ ID NOs: 63 and 64 and detected withprobes indicated as SEQ ID NOs: 162 and 163. In the second haplotypewindow, the SNP marker used to monitor the introgression of ASRresistance locus 10, ASR resistance locus 11, ASR resistance locus 12,and ASR resistance locus 13 is NS0124935. This SNP marker DNA sequences(presented as SEQ ID NO: 99) can be amplified using the primersindicated as SEQ ID NOs: 65 and 66 and detected with probes indicated asSEQ ID NOs: 164 and 165.

It is further understood, that one or more markers mapped within 10centimorgans or less from said marker molecules can be used for theselection and introgression of ASR resistance loci.

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising theagents of the present invention.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules are exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., (1989) at9.47-9.52, 9.56-9.58, Kanehisa Nucl. Acids Res. 12:203-213, (1984), andWetmur et al., J. Mol. Biol. 31:349-370 (1968). Appropriate stringencyconditions that promote DNA hybridization are, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,1989, 6.3.1-6.3.6. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

For example, hybridization using DNA or RNA probes or primers can beperformed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.Detection of DNA segments via hybridization is well-known to those ofskill in the art, and thus depending on the application envisioned, onewill desire to employ varying hybridization conditions to achievevarying degrees of selectivity of probe towards target sequence and themethod of choice will depend on the desired results.

As used herein, an agent, be it a naturally occurring molecule orotherwise may be “substantially purified”, if desired, referring to amolecule separated from substantially all other molecules normallyassociated with it in its native state. More preferably a substantiallypurified molecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free, and most preferably 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The term “substantially purified” is not intended to encompassmolecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal., Science 238:336-340 (1987), European Patent 144914), chemicallabels (U.S. Pat. No. 4,582,789, U.S. Pat. No. 4,563,417), modifiedbases (European Patent 119448), all of which are herein incorporated byreference in their entirety).

In a preferred embodiment, a nucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO: 67 through SEQ ID NO: 99 or complements thereof orfragments of either under moderately stringent conditions, for exampleat about 2.0×SSC and about 65° C. In a particularly preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 67 through SEQ ID NO: 99 or complements or fragments of eitherunder high stringency conditions. In one aspect of the presentinvention, a preferred marker nucleic acid molecule of the presentinvention has the nucleic acid sequence set forth in SEQ ID NO: 67through SEQ ID NO: 99 or complements thereof or fragments of either. Inanother aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with the nucleic acid sequence set forth in SEQID NO: 67 through SEQ ID NO: 99 or complement thereof or fragments ofeither. In a further aspect of the present invention, a preferred markernucleic acid molecule of the present invention shares between 95% and100% sequence identity with the sequence set forth in SEQ ID NO: 67through SEQ ID NO: 99 or complement thereof or fragments of either. In amore preferred aspect of the present invention, a preferred markernucleic acid molecule of the present invention shares between 98% and100% sequence identity with the nucleic acid sequence set forth in SEQID NO: 67 through SEQ ID NO: 99 or complement thereof or fragments ofeither.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with fungal disease resistance of soybean of thepresent invention. Examples of public marker databases include, forexample: Soybase, an Agricultural Research Service, United StatesDepartment of Agriculture.

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations where individuals are predominantly homozygous and loci arepredominantly dimorphic, dominant and codominant markers can be equallyvaluable. As populations become more heterozygous and multiallelic,codominant markers often become more informative of the genotype thandominant markers.

Markers, such as simple sequence repeat markers (SSR), AFLP markers,RFLP markers, RAPD markers, phenotypic markers, SNPs, isozyme markers,microarray transcription profiles that are genetically linked to orcorrelated with alleles of a QTL of the present invention can beutilized (Walton, Seed World 22-29 (July, 1993), Burow et al., MolecularDissection of Complex Traits, 13-29, ed. Paterson, CRC Press, New York(1988)). Methods to isolate such markers are known in the art. Forexample, locus-specific SSR markers can be obtained by screening agenomic library for microsatellite repeats, sequencing of “positive”clones, designing primers which flank the repeats, and amplifyinggenomic DNA with these primers. The size of the resulting amplificationproducts can vary by integral numbers of the basic repeat unit. Todetect a polymorphism, PCR products can be radiolabeled, separated ondenaturing polyacrylamide gels, and detected by autoradiography.Fragments with size differences >4 bp can also be resolved on agarosegels, thus avoiding radioactivity.

The detection of polymorphic sites in a sample of DNA, RNA, or cDNA maybe facilitated through the use of nucleic acid amplification methods.Such methods specifically increase the concentration of polynucleotidesthat span the polymorphic site, or include that site and sequenceslocated either distal or proximal to it. Such amplified molecules can bereadily detected by gel electrophoresis or other means.

The most preferred method of achieving such amplification employs thepolymerase chain reaction (PCR) (Mullis et al., Cold Spring Harbor Symp.Quant. Biol. 51:263-273 (1986), European Patent Appln. 50,424, EuropeanPatent 84,796, European Patent 258,017, European Patent 237,362,European Patent 201,184, U.S. Pat. No. 4,683,202, U.S. Pat. No.4,582,788, U.S. Pat. No. 4,683,194), using primer pairs that are capableof hybridizing to the proximal sequences that define a polymorphism inits double-stranded form.

In lieu of PCR, alternative methods, such as the “Ligase Chain Reaction”(LCR) may be used (Barany, Proc. Natl. Acad. Sci. (USA.) 88:189-193(1991), the entirety of which is herein incorporated by reference). LCRuses two pairs of oligonucleotide probes to exponentially amplify aspecific target. The sequence of each pair of oligonucleotides isselected to permit the pair to hybridize to abutting sequences of thesame strand of the target. Such hybridization forms a substrate for atemplate-dependent ligase. As with PCR, the resulting products thusserve as a template in subsequent cycles and an exponentialamplification of the desired sequence is obtained.

The “Oligonucleotide Ligation Assay” (OLA) may alternatively be employed(Landegren et al., Science 241:1077-1080 (1988), the entirety of whichis herein incorporated by reference). The OLA protocol uses twooligonucleotides that are designed to be capable of hybridizing toabutting sequences of a single strand of a target. OLA, like LCR, isparticularly suited for the detection of point mutations. Unlike LCR,however, OLA results in “linear” rather than exponential amplificationof the target sequence.

Schemes based on ligation of two (or more) oligonucleotides in thepresence of a nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu et al., Genomics 4:560-569 (1989), the entirety of whichis herein incorporated by reference), and may be readily adapted to thepurposes of the present invention.

Other known nucleic acid amplification procedures, such asallele-specific oligomers, branched DNA technology, transcription-basedamplification systems, or isothermal amplification methods may also beused to amplify and analyze such polymorphisms (U.S. Pat. No. 5,130,238,European Patent 329,822, U.S. Pat. No. 5,169,766, European Patent359,789, Kwoh, et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173-1177(1989) European Patent 368,906, Walker et al., Proc. Natl. Acad. Sci.(USA.) 89:392-396 (1992), all of which are herein incorporated byreference in their entirety).

Polymorphisms can also be identified by Single Strand ConformationPolymorphism (SSCP) analysis. SSCP is a method capable of identifyingmost sequence variations in a single strand of DNA, typically between150 and 250 nucleotides in length (Elles, Methods in Molecular Medicine:Molecular Diagnosis of Genetic Diseases, Humana Press (1996); Orita etal., Genomics 5: 874-879 (1989)). Under denaturing conditions a singlestrand of DNA will adopt a conformation that is uniquely dependent onits sequence conformation. This conformation usually will be different,even if only a single base is changed. Most conformations have beenreported to alter the physical configuration or size sufficiently to bedetectable by electrophoresis.

A central attribute of SNPs is that the site of the polymorphism is at asingle nucleotide. SNPs are more stable than other classes ofpolymorphisms. Their spontaneous mutation rate is approximately 10⁻⁹(Kornberg, DNA Replication, W. H. Freeman & Co., San Francisco (1980)).As SNPs result from sequence variation, new polymorphisms can beidentified by sequencing random genomic or cDNA molecules. SNPs can alsoresult from deletions, point mutations and insertions. That said, SNPsare also advantageous as markers since they are often diagnostic of“identity by descent” because they rarely arise from independentorigins. Any single base alteration, whatever the cause, can be a SNP.SNPs occur at a greater frequency than other classes of polymorphismsand can be more readily identified. In the present invention, a SNP canrepresent a single indel event, which may consist of one or more basepairs, or a single nucleotide polymorphism.

SNPs can be characterized using any of a variety of methods. Suchmethods include the direct or indirect sequencing of the site, the useof restriction enzymes where the respective alleles of the site createor destroy a restriction site, the use of allele-specific hybridizationprobes, the use of antibodies that are specific for the proteins encodedby the different alleles of the polymorphism, or by other biochemicalinterpretation. SNPs can be sequenced using a variation of the chaintermination method (Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.) 74:5463-5467 (1977)) in which the use of radioisotopes are replaced withfluorescently-labeled dideoxy nucleotides and subjected to capillarybased automated sequencing (U.S. Pat. No. 5,332,666, the entirety ofwhich is herein incorporated by reference; U.S. Pat. No. 5,821,058, theentirety of which is herein incorporated by reference). Automatedsequencers are available from, for example, Applied Biosystems, FosterCity, Calif. (3730xl DNA Analyzer), Beckman Coulter, Fullerton, Calif.(CEQ™ 8000 Genetic Analysis System) and LI-COR, Inc., Lincoln, Nebr.(4300 DNA Analysis System).

Approaches for analyzing SNPs can be categorized into two groups. Thefirst group is based on primer-extension assays, such as solid-phaseminisequencing or pyrosequencing. In the solid-phase minisequencingmethod, a DNA polymerase is used specifically to extend a primer thatanneals immediately adjacent to the variant nucleotide. A single labelednucleoside triphospate complementary to the nucleotide at the variantsite is used in the extension reaction. Only those sequences thatcontain the nucleotide at the variant site will be extended by thepolymerase. A primer array can be fixed to a solid support wherein eachprimer is contained in four small wells, each well being used for one ofthe four nucleoside triphospates present in DNA. Template DNA or RNAfrom each test organism is put into each well and allowed to anneal tothe primer. The primer is then extended one nucleotide using apolymerase and a labeled di-deoxy nucleotide triphosphate. The completedreaction can be imaged using devices that are capable of detecting thelabel which can be radioactive or fluorescent. Using this method severaldifferent SNPs can be visualized and detected (Syvänen et al., Hum.Mutat. 13: 1-10 (1999)). The pyrosequencing technique is based on anindirect bioluminometric assay of the pyrophosphate (PPi) that isreleased from each dNTP upon DNA chain elongation. Following Klenowpolymerase mediated base incorporation, PPi is released and used as asubstrate, together with adenosine 5-phosphosulfate (APS), for ATPsulfurylase, which results in the formation of ATP. Subsequently, theATP accomplishes the conversion of luciferin to its oxi-derivative bythe action of luciferase. The ensuing light output becomes proportionalto the number of added bases, up to about four bases. To allowprocessivity of the method dNTP excess is degraded by apyrase, which isalso present in the starting reaction mixture, so that only dNTPs areadded to the template during the sequencing procedure (Alderborn et al.,Genome Res. 10: 1249-1258 (2000)). An example of an instrument designedto detect and interpret the pyrosequencing reaction is available fromBiotage, Charlottesville, Va. (PyroMark MD).

A more recent SNP detection method, based on primer-extension assays isthe GOOD assay. The GOOD assay (Sauer et al., Nucleic Acids Res. 28:e100 (2000)) is an allele-specific primer extension protocol thatemploys MALDI-TOF (matrix-assisted laser desorption/ionizationtime-of-flight) mass spectrometry. The region of DNA containing a SNP isamplified first by PCR amplification. Residual dNTPs are destroyed usingan alkaline phosphatase. Allele-specific products are then generatedusing a specific primer, a conditioned set of a-S-dNTPs and a-S-ddNTPsand a fresh DNA polymerase in a primer extension reaction. UnmodifiedDNA is removed by 5′phosphodiesterase digestion and the modifiedproducts are alkylated to increase the detection sensitivity in the massspectrometric analysis. All steps are carried out in a single vial atthe lowest practical sample volume and require no purification. Theextended reaction can be given a positive or negative charge and isdetected using mass spectrometry (Sauer et al., Nucleic Acids Res. 28:e13 (2000)). An instrument in which the GOOD assay is analyzed is forexample, the AUTOFLEX® MALDI-TOF system from Bruker Daltonics(Billerica, Mass.).

The second group, which is based on recognition of heteroduplex DNAmolecules, includes oligonucleotide hybridization, TAQ-MAN® assays,molecular beacons, electronic dot blot assays and denaturinghigh-performance liquid chromatography. Oligonucleotide hybridizationscan be performed in mass using micro-arrays (Southern, Trends Genet. 12:110-115 (1996)). TAQ-MAN® assays, or Real Time PCR, detects theaccumulation of a specific PCR product by hybridization and cleavage ofa double-labeled fluorogenic probe during the amplification reaction. ATAQ-MAN® assay includes four oligonucleotides, two of which serve as PCRprimers and generate a PCR product encompassing the polymorphism to bedetected. The other two are allele-specificfluorescence-resonance-energy-transfer (FRET) probes. FRET probesincorporate a fluorophore and a quencher molecule in close proximity sothat the fluorescence of the fluorophore is quenched. The signal from aFRET probes is generated by degradation of the FRET oligonucleotide, sothat the fluorophore is released from proximity to the quencher, and isthus able to emit light when excited at an appropriate wavelength. Inthe assay, two FRET probes bearing different fluorescent reporter dyesare used, where a unique dye is incorporated into an oligonucleotidethat can anneal with high specificity to only one of the two alleles.Useful reporter dyes include 6-carboxy-4,7,2′,7′-tetrachlorofluorecein(TET), 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and6-carboxyfluorescein phosphoramidite (FAM). A useful quencher is6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA). Annealed (but notnon-annealed) FRET probes are degraded by TAQ DNA polymerase as theenzyme encounters the 5′ end of the annealed probe, thus releasing thefluorophore from proximity to its quencher. Following the PCR reaction,the fluorescence of each of the two fluorescers, as well as that of thepassive reference, is determined fluorometrically. The normalizedintensity of fluorescence for each of the two dyes will be proportionalto the amounts of each allele initially present in the sample, and thusthe genotype of the sample can be inferred. An example of an instrumentused to detect the fluorescence signal in TAQ-MAN® assays, or Real TimePCR are the 7500 Real-Time PCR System (Applied Biosystems, Foster City,Calif.).

Molecular beacons are oligonucleotide probes that form a stem-and-loopstructure and possess an internally quenched fluorophore. When they bindto complementary targets, they undergo a conformational transition thatturns on their fluorescence. These probes recognize their targets withhigher specificity than linear probes and can easily discriminatetargets that differ from one another by a single nucleotide. The loopportion of the molecule serves as a probe sequence that is complementaryto a target nucleic acid. The stem is formed by the annealing of the twocomplementary arm sequences that are on either side of the probesequence. A fluorescent moiety is attached to the end of one arm and anonfluorescent quenching moiety is attached to the end of the other arm.The stem hybrid keeps the fluorophore and the quencher so close to eachother that the fluorescence does not occur. When the molecular beaconencounters a target sequence, it forms a probe-target hybrid that isstronger and more stable than the stem hybrid. The probe undergoesspontaneous conformational reorganization that forces the arm sequencesapart, separating the fluorophore from the quencher, and permitting thefluorophore to fluoresce (Bonnet et al., 1999). The power of molecularbeacons lies in their ability to hybridize only to target sequences thatare perfectly complementary to the probe sequence, hence permittingdetection of single base differences (Kota et al., Plant Mol. Biol. Rep.17: 363-370 (1999)). Molecular beacon detection can be performed forexample, on the Mx4000® Multiplex Quantitative PCR System fromStratagene (La Jolla, Calif.).

The electronic dot blot assay uses a semiconductor microchip comprisedof an array of microelectrodes covered by an agarose permeation layercontaining streptavidin. Biotinylated amplicons are applied to the chipand electrophoresed to selected pads by positive bias direct current,where they remain embedded through interaction with streptavidin in thepermeation layer. The DNA at each pad is then hybridized to mixtures offluorescently labeled allele-specific oligonucleotides. Single base pairmismatched probes can then be preferentially denatured by reversing thecharge polarity at individual pads with increasing amperage. The arrayis imaged using a digital camera and the fluorescence quantified as theamperage is ramped to completion. The fluorescence intensity is thendetermined by averaging the pixel count values over a region of interest(Gilles et al., Nature Biotech. 17: 365-370 (1999)).

A more recent application based on recognition of heteroduplex DNAmolecules uses denaturing high-performance liquid chromatography(DHPLC). This technique represents a highly sensitive and fullyautomated assay that incorporates a Peltier-cooled 96-well autosamplerfor high-throughput SNP analysis. It is based on an ion-pairreversed-phase high performance liquid chromoatography method. The heartof the assay is a polystyrene-divinylbenzene copolymer, which functionsas a stationary phase. The mobile phase is composed of an ion-pairingagent, triethylammonium acetate (TEAA) buffer, which mediates thebinding of DNA to the stationary phase, and an organic agent,acetonitrile (ACN), to achieve subsequent separation of the DNA from thecolumn. A linear gradient of CAN allows the separation of fragmentsbased on the presence of heteroduplexes. DHPLC thus identifies mutationsand polymorphisms that cause heteroduplex formation between mismatchednucleotides in double-stranded PCR-amplified DNA. In a typical assay,sequence variation creates a mixed population of heteroduplexes andhomoduplexes during reannealing of wild-type and mutant DNA. When thismixed population is analyzed by DHPLC under partially denaturingtemperatures, the heteroduplex molecules elute from the column prior tothe homoduplex molecules, because of their reduced melting temperatures(Kota et al., Genome 44: 523-528 (2001)). An example of an instrumentused to analyze SNPs by DHPLC is the WAVE® HS System from Transgenomic,Inc. (Omaha, Nebr.).

A microarray-based method for high-throughput monitoring of plant geneexpression can be utilized as a genetic marker system. This ‘chip’-basedapproach involves using microarrays of nucleic acid molecules asgene-specific hybridization targets to quantitatively or qualitativelymeasure expression of plant genes (Schena et al., Science 270:467-470(1995), the entirety of which is herein incorporated by reference;Shalon, Ph.D. Thesis. Stanford University (1996), the entirety of whichis herein incorporated by reference). Every nucleotide in a largesequence can be queried at the same time. Hybridization can be used toefficiently analyze nucleotide sequences. Such microarrays can be probedwith any combination of nucleic acid molecules. Particularly preferredcombinations of nucleic acid molecules to be used as probes include apopulation of mRNA molecules from a known tissue type or a knowndevelopmental stage or a plant subject to a known stress (environmentalor man-made) or any combination thereof (e.g. mRNA made from waterstressed leaves at the 2 leaf stage). Expression profiles generated bythis method can be utilized as markers.

For the purpose of QTL mapping, the markers included must be diagnosticof origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is very low. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander and Botstein, Genetics, 121:185-199 (1989), andthe interval mapping, based on maximum likelihood methods described byLander and Botstein, Genetics, 121:185-199 (1989), and implemented inthe software package MAPMAKER/QTL (Lincoln and Lander, Mapping GenesControlling Quantitative Traits Using MAPMAKER/QTL, Whitehead Institutefor Biomedical Research, Massachusetts, (1990). Additional softwareincludes Qgene, Version 2.23 (1996), Department of Plant Breeding andBiometry, 266 Emerson Hall, Cornell University, Ithaca, N.Y., the manualof which is herein incorporated by reference in its entirety). Use ofQgene software is a particularly preferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander andBotstein, Genetics, 121:185-199 (1989), and further described by Arúsand Moreno-Gonzalez, Plant Breeding, Hayward, Bosemark, Romagosa (eds.)Chapman & Hall, London, pp. 314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak and Lander, Genetics, 139:1421-1428(1995), the entirety of which is herein incorporated by reference).Multiple regression methods or models can be also be used, in which thetrait is regressed on a large number of markers (Jansen, Biometrics inPlant Breed, van Oijen, Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 116-124 (1994); Weber and Wricke, Advances in Plant Breeding,Blackwell, Berlin, 16 (1994)). Procedures combining interval mappingwith regression analysis, whereby the phenotype is regressed onto asingle putative QTL at a given marker interval, and at the same timeonto a number of markers that serve as ‘cofactors,’ have been reportedby Jansen and Stam, Genetics, 136:1447-1455 (1994) and Zeng, Genetics,136:1457-1468 (1994). Generally, the use of cofactors reduces the biasand sampling error of the estimated QTL positions (Utz and Melchinger,Biometrics in Plant Breeding, van Oijen, Jansen (eds.) Proceedings ofthe Ninth Meeting of the Eucarpia Section Biometrics in Plant Breeding,The Netherlands, pp. 195-204 (1994), thereby improving the precision andefficiency of QTL mapping (Zeng, Genetics, 136:1457-1468 (1994)). Thesemodels can be extended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al., Theo. Appl. Genet.91:33-37 (1995).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingof plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (eds.). Plenum Press, New York,pp. 157-173 (1988), the entirety of which is herein incorporated byreference). Consideration must be given to the source of parents(adapted vs. exotic) used in the mapping population. Chromosome pairingand recombination rates can be severely disturbed (suppressed) in widecrosses (adapted×exotic) and generally yield greatly reduced linkagedistances. Wide crosses will usually provide segregating populationswith a relatively large array of polymorphisms when compared to progenyin a narrow cross (adapted×adapted).

An F₂ population is the first generation of selfing after the hybridseed is produced. Usually a single F₁ plant is selfed to generate apopulation segregating for all the genes in Mendelian (1:2:1) fashion.Maximum genetic information is obtained from a completely classified F₂population using a codominant marker system (Mather, Measurement ofLinkage in Heredity: Methuen and Co., (1938), the entirety of which isherein incorporated by reference). In the case of dominant markers,progeny tests (e.g F₃, BCF₂) are required to identify the heterozygotes,thus making it equivalent to a completely classified F₂ population.However, this procedure is often prohibitive because of the cost andtime involved in progeny testing. Progeny testing of F₂ individuals isoften used in map construction where phenotypes do not consistentlyreflect genotype (e.g. disease resistance) or where trait expression iscontrolled by a QTL. Segregation data from progeny test populations(e.g. F₃ or BCF₂) can be used in map construction. Marker-assistedselection can then be applied to cross progeny based on marker-trait mapassociations (F₂, F₃), where linkage groups have not been completelydisassociated by recombination events (i.e., maximum disequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al., Proc. Natl. Acad. Sci.(USA.) 89:1477-1481 (1992)). However, as the distance between markersbecomes larger (i.e., loci become more independent), the information inRIL populations decreases dramatically when compared to codominantmarkers.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al., Proc. Natl. Acad. Sci. (USA.) 89:1477-1481 (1992)). Informationobtained from backcross populations using either codominant or dominantmarkers is less than that obtained from F₂ populations because one,rather than two, recombinant gametes are sampled per plant. Backcrosspopulations, however, are more informative (at low marker saturation)when compared to RILs as the distance between linked loci increases inRIL populations (i.e. about 0.15% recombination). Increasedrecombination can be beneficial for resolution of tight linkages, butmay be undesirable in the construction of maps with low markersaturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore, et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832(1991)). In BSA, two bulked DNA samples are drawn from a segregatingpopulation originating from a single cross. These bulks containindividuals that are identical for a particular trait (resistant orsusceptible to particular disease) or genomic region but arbitrary atunlinked regions (i.e. heterozygous). Regions unlinked to the targetregion will not differ between the bulked samples of many individuals inBSA.

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.2006 Genetics). This approach tracks blocks of DNA known as haplotypes,as defined by polymorphic markers, which are assumed to be identical bydescent in the mapping population. This assumption results in a largereffective sample size, offering greater resolution of QTL. Methods fordetermining the statistical significance of a correlation between aphenotype and a genotype, in this case a haplotype, may be determined byany statistical test known in the art and with any accepted threshold ofstatistical significance being required. The application of particularmethods and thresholds of significance are well with in the skill of theordinary practitioner of the art.

The SNP markers of the present invention can be used to isolate orsubstantially purify an allele of a QTL that is also located on linkagegroup associated with ASR resistance locus 1, ASR resistance locus 2,ASR resistance locus 3, ASR resistance locus 4, ASR resistance locus 5,ASR resistance locus 6, ASR resistance locus 7, ASR resistance locus 8,ASR resistance locus 9, ASR resistance locus 10, ASR resistance locus11, ASR resistance locus 12, and ASR resistance locus 13. Constructionof an overlapping series of clones (a clone contig) across the regioncan provide the basis for a physical map encompassing an allele of afungal disease resistance QTL that are located on a linkage groupassociated with ASR resistance locus 1, ASR resistance locus 2, ASRresistance locus 3, ASR resistance locus 4, ASR resistance locus 5, ASRresistance locus 6, ASR resistance locus 7, ASR resistance locus 8, ASRresistance locus 9, ASR resistance locus 10, ASR resistance locus 11,ASR resistance locus 12, and ASR resistance locus 13. The yeastartificial chromosome (YAC) cloning system has facilitated chromosomewalking and large-size cloning strategies. A sequence tag site (STS)content approach utilizing the markers of the present invention can beused for the construction of YAC clones across chromosome regions. Suchan STS content approach to the construction of YAC maps can provide adetailed and ordered STS-based map of any chromosome region, includingthe region encompassing the allele of a QTL is also located on a linkagegroup associated with ASR resistance locus 1, ASR resistance locus 2,ASR resistance locus 3, ASR resistance locus 4, ASR resistance locus 5,ASR resistance locus 6, ASR resistance locus 7, ASR resistance locus 8,ASR resistance locus 9, ASR resistance locus 10, ASR resistance locus11, ASR resistance locus 12, and ASR resistance locus 13. YAC maps canbe supplemented by detailed physical maps are constructed across theregion by using BAC, PAC, or bacteriophage P1 clones that containinserts ranging in size from 70 kb to several hundred kilobases (Cregan,Theor. Appl. Gen. 78:919-928 (1999), Sternberg, Proc. Natl. Acad. Sci.87:103-107 (1990), Sternberg, Trends Genet. 8:11-16 (1992); Sternberg etal., New Biol. 2:151-162 (1990); Ioannou et al., Nat. Genet. 6:84-89(1994); Shizuya et al., Proc. Natl. Acad. Sci. 89: 8794-8797 (1992), allof which are herein incorporated by reference in their entirety).

Overlapping sets of clones are derived by using the available markers ofthe present invention to screen BAC, PAC, bacteriophage P1, or cosmidlibraries. In addition, hybridization approaches can be used to convertthe YAC maps into BAC, PAC, bacteriophage P1, or cosmid contig maps.Entire YACs and products of inter-A/u-PCR as well as primer sequencesfrom appropriate STSs can be used to screen BAC, PAC, bacteriophage P1,or cosmid libraries. The clones isolated for any region can be assembledinto contigs using STS content information and fingerprinting approaches(Sulston et al., Comput. Appl. Biosci. 4:125-132 (1988)).

The degeneracy of the genetic code, which allows different nucleic acidsequences to code for the same protein or peptide, is known in theliterature. As used herein a nucleic acid molecule is degenerate ofanother nucleic acid molecule when the nucleic acid molecules encode forthe same amino acid sequences but comprise different nucleotidesequences. An aspect of the present invention is that the nucleic acidmolecules of the present invention include nucleic acid molecules thatare degenerate of the nucleic acid molecule that encodes the protein(s)of the quantitative trait alleles.

Another aspect of the present invention is that the nucleic acidmolecules of the present invention include nucleic acid molecules thatare homologues of the nucleic acid molecule that encodes the one or moreof the proteins associated with the QTL.

Exogenous genetic material may be transferred into a plant by the use ofa DNA plant transformation vector or construct designed for such apurpose. A particularly preferred subgroup of exogenous materialcomprises a nucleic acid molecule of the present invention. Design ofsuch a vector is generally within the skill of the art (See, PlantMolecular Biology: A Laboratory Manual, eds. Clark, Springer, New York(1997), Examples of such plants, include, without limitation, alfalfa,Arabidopsis, barley, Brassica, broccoli, cabbage, citrus, cotton,garlic, oat, oilseed rape, onion, canola, flax, maize, an ornamentalplant, pea, peanut, pepper, potato, rice, rye, sorghum, soybean,strawberry, sugarcane, sugarbeet, tomato, wheat, poplar, pine, fir,eucalyptus, apple, lettuce, lentils, grape, banana, tea, turf grasses,sunflower, oil palm, Phaseolus etc.

A construct or vector may include the endogenous promoter of the fungaldisease resistance QTL of the present invention. The characteristic offungal disease resistance might best be achieved by expressing theidentified QTL protein with the endogenous promoter. Alternatively, aheterologous promoter may be selected to express the protein or proteinfragment of choice. These promoters may be operably linked to apolynucleotide sequence encoding the protein corresponding to the fungalresistance QTL. The heterologous promoter may be one that is selectedbased upon maturation or flowering time, in that timing of expression ofthe desired protein may be critical to the parameters affecting thefungal disease resistance trait. Effective expression of the fungaldisease resistance QTL may require promoters that express in specifictissue types as well.

Alternatively, the promoters may be operably linked to other nucleicacid sequences, such as those encoding transit peptides, selectablemarker proteins, or antisense sequences. The promoters may be selectedon the basis of the cell type into which the vector will be inserted oron the basis of its regulatory features. Examples of such featuresinclude enhancement of transcriptional activity, inducibility,tissue-specificity, and developmental stage-specificity. In plants,promoters that are inducible, of viral or synthetic origin,constitutively active, temporally regulated, and spatially regulatedhave been described (Poszkowski, et al., EMBO J., 3: 2719, 1989; Odell,et al., Nature, 313:810, 1985; Chau et al., Science, 244:174-181. 1989).Often-used constitutive promoters include the CaMV 35S promoter (Odell,et al., Nature, 313: 810, 1985), the enhanced CaMV 35S promoter, theFigwort Mosaic Virus (FMV) promoter (Richins, et al., Nucleic Acids Res.20: 8451, 1987), the nopaline synthase (nos) promoter (Shaw et al.,Nucleic Acids Res. 12: 7831-7846 (1984)) and the octopine synthase (ocs)promoter.

Useful inducible promoters include promoters induced by salicylic acidor polyacrylic acids (PR-1; Williams, et al., Biotechnology 10:540-543,1992), induced by application of safeners (substitutedbenzenesulfonamide herbicides; Hershey and Stoner, Plant Mol. Biol. 17:679-690, 1991), heat-shock promoters (Ou-Lee et al., Proc. Natl. Acad.Sci U.S.A. 83: 6815, 1986; Ainley et al., Plant Mol. Biol. 14: 949,1990), a nitrate-inducible promoter derived from the spinach nitritereductase transcribable polynucleotide sequence (Back et al., Plant Mol.Biol. 17: 9, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki etal., Plant Mol. Biol. 15: 905, 1990), and light-inducible promotersassociated with the small subunit of RuBP carboxylase and LHCP families(Kuhlemeier et al., Plant Cell 1: 471, 1989; Feinbaum et al., Mol. Gen.Genet. 226: 449-456, 1991; Weisshaar, et al., EMBO J. 10: 1777-1786,1991; Lam and Chua, J. Biol. Chem. 266: 17131-17135, 1990; Castresana etal., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert, et al., EMBO J. 8: 651,1989).

Particularly preferred promoters in the recombinant vector include thenopaline synthase (NOS) promoter (Ebert et al., 1987), the octopinesynthase (OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), theCaMV 35S promoter (Odell et al., 1985), the figwort mosaic virus35S-promoter (Walker et al., 1987); the light-inducible promoter fromthe small subunit of ribulose-1,5-bisphosphate carboxylase (ssRUBlSCO);the EIF-4A promoter from tobacco (Mandel, et al., Plant Mol. Biol, 29:995-1004, 1995); the chitinase promoter from Arabidopsis (Samac, et al.,Plant Cell, 3:1063-1072, 1991); the LTP (Lipid Transfer Protein)promoters from broccoli (Pyee, et al., Plant J., 7: 49-59, 1995);petunia chalcone isomerase (Van Tunen, et al., EMBO J. 7: 1257, 1988);bean glycine rich protein 1 (Keller, et al., EMBO L., 8: 1309-1314,1989); the Potato patatin (Wenzler, et al., Plant Mol. Biol., 12: 41-50,1989); the Arabidopsis Actin 7 promoter (GENBANK accession U27811.1GI:1002528, 17 Apr. 1997 and PCT application: WO0144457A2; the entiretyof which is herein incorporated by reference); the Arabidopsis Actin 8promoter (An et al., Plant J. 10: 107-121 (1996) and PCT application:WO0144457A2); the Arabidopsis Rubisco small subunit 4 promoter (Krebberset al., Plant Mol. Biol. 11: 745-759 (1988)); the Brassica napin genepromoter (U.S. Pat. No. 5,420,034, the entirety of which is hereinincorporated by reference); the Arabidopsis Suc2 promoter (Truemit etal., Planta 196: 564-570 (1995)); Arabidopsis elongation factor EF-1alpha promoter (Axelos et al., Mol. Gen. Genet. 219: 106-112 (1989));and the Glycine max 7sα beta conglycin promoter, Sphas (Doyle et al., J.Biol. Chem. 261: 9228-9238 (1986)).

Constructs of the present invention may also include additional 5′untranslated regions (5′ UTR) or leaders of an mRNA polynucleotidemolecule or gene which can play an important role in translationinitiation. Some 5′ UTRs may act as translational enhancers and may alsobe incorporated as part of the recombinant vector. For example,non-translated 5′ leader polynucleotide molecules derived from heatshock protein genes have been demonstrated to enhance gene expression inplants (see for example, U.S. Pat. No. 5,659,122, the entirety of whichis herein incorporated by reference and U.S. Pat. No. 5,362,865, theentirety of which is herein incorporated by reference). Thus therecombinant vector may preferably contain one or more 5′ non-translatedleader sequences which serve to enhance expression of the nucleic acidsequence. Such enhancer sequences may be desirable to increase or alterthe translational efficiency of the resultant mRNA. Preferred 5′ nucleicacid sequences include the Arabidopsis Actin 7 leader (GENBANK accessionU27811.1 GI:1002528, 17 Apr. 1997 and PCT application: WO0144457A2; theentirety of which is herein incorporated by reference); the ArabidopsisActin 8 leader (An et al., Plant J. 10: 107-121 (1996) and PCTapplication: WO0144457A2); the Arabidopsis Rubisco small subunit 4leader (Krebbers et al., Plant Mol. Biol. 11: 745-759 (1988)); theBrassica napin gene leader (U.S. Pat. No. 5,420,034, the entirety ofwhich is herein incorporated by reference); the Arabidopsis Suc2 leader(Truernit et al., Planta 196: 564-570 (1995)); the Petunia hybrida Hsp70gene leader (Winter et al., Mol. Gen. Genet. 211: 315-319 (1988)): theArabidopsis EPSPS gene leader (Klee et al., Mol. Gen. Genet. 210:437-442 (1987)); the Arabidopsis elongation factor EF-1 alpha leader(Axelos et al., Mol. Gen. Genet. 219: 106-112 (1989)); and the Glycinemax 7sα beta conglycin leader (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)). These additional upstream regulatory polynucleotidemolecules may be derived from a source that is native or heterologouswith respect to the other elements present on the construct.

In addition, constructs may include additional regulatory polynucleotidemolecules from the 3′-untranslated region (3′ UTR) of plant genes. A 3′UTR or terminator typically provides a transcriptional terminationsignal, and a polyadenylation signal which functions in plants to causethe addition of adenylate nucleotides to the 3′ end of the mRNA.Usually, nucleic acid sequences located a few hundred base pairsdownstream of the polyadenylation site serve to terminate transcription.In addition, some 3′ UTRs provide additional properties such asenhancing the stability of the mRNA as in the potato proteinaseinhibitor II gene 3′ UTR (An et al., The Plant Cell 1: 115-122 (1989)).Other 3′ UTRs may provide sequences that enhance degredation of the mRNAsuch as the 5′-UUAUUUAUU-3′ motif shown to contribute to lower stabilityof RNA messages in animal cells (Zubiaga et al., Mol. Cell Biol. 15:2219-2230 (1995)). These additional downstream regulatory polynucleotidemolecules may be derived from a source that is native or heterologouswith respect to the other elements present on the construct.

Preferred 3′ UTRs or terminators are the potato proteinase inhibitor IIgene 3′ UTR (An et al., The Plant Cell 1: 115-122 (1989)); the peaRubisco small subunit E9 terminator (Coruzzi et al., EMBO J. 3:1671-1679 (1984)); the cauliflower mosaic virus 35S terminator; theBrassica napin gene terminator (U.S. Pat. No. 5,420,034); the Glycinemax 7sα beta conglycin gene terminator (Doyle et al., J. Biol. Chem.261: 9228-9238 (1986)); the Phaseoulus vulgaris Arc5 terminator(Goossens et al., Eur. J. Biochem. 225: 787-795 (1994)); theAgrobacterium tumefaciens nopaline synthase terminator (Rojiyaa et al.,1987, GENBANK Accession E01312 and U.S. Patent ApplicationUS20020192813A1, the entirety of which is herein incorporated byreference); and the Glycine max ADR12 gene terminator (Datta et al.,Plant Mol. Biol. 21: 859-869 (1993)).

A vector or construct may also include regulatory elements derived fromthe introns of certain genes. Examples of such include the Adh intron 1(Callis et al., Genes and Develop. 1:1183-1200 (1987); the sucrosesynthase intron (Vasil et al., Plant Physiol. 91:1575-1579 (1989); andthe TMV omega element (Gallie et al., The Plant Cell 1:301-311 (1989)).Preferred introns are the Arabidopsis Actin 7 intron (GENBANK accessionU27811.1 GI:1002528, 17 Apr. 1997 and PCT application: WO200144457A2;the entirety of which is herein incorporated by reference); theArabidopsis Actin 8 intron (An et al., Plant J. 10: 107-121 (1996) andPCT application: WO200144457A2); and the Arabidopsis elongation factorEF-1 alpha inton (Axelos et al., Mol. Gen. Genet. 219: 106-112 (1989))These and other regulatory elements may be included when appropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet.199:183-188 (1985)), which codes for kanamycin resistance and can beselected for using kanamycin, G418, etc.; a bar gene which codes forbialaphos resistance; a mutant EPSP synthase gene (Hinchee et al.,Bio/Technology 6:915-922 (1988)), which encodes glyphosate resistance; anitrilase gene which confers resistance to bromoxynil (Stalker et al.,J. Biol. Chem. 263:6310-6314 (1988)); a mutant acetolactate synthasegene (ALS) which confers imidazolinone or sulphonylurea resistance (forexample, U.S. Pat. No. 6,222,100, the entirety of which is hereinincorporated by reference); a methotrexate resistant DHFR gene (Thilletet al., J. Biol. Chem. 263:12500-12508 (1988)); Dicamba toleranceconferred, for example, by a gene for dicamba monooxygenase (DMO) fromPseudomonas maltophilia (US Patent Application 20030135879, the entiretyof which is herein incorporated by reference).

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude a β-glucuronidase or uidA gene (GUS) which encodes an enzyme forwhich various chromogenic substrates are known (Jefferson, Plant Mol.Biol, Rep. 5:387-405 (1987), the entirety of which is hereinincorporated by reference; Jefferson et al., EMBO J. 6:3901-3907 (1987),the entirety of which is herein incorporated by reference); an R-locusgene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta et al.,Stadler Symposium 11:263-282 (1988), the entirety of which is hereinincorporated by reference); a β-lactamase gene (Sutcliffe et al., Proc.Natl. Acad. Sci. (USA.) 75:3737-3741 (1978), the entirety of which isherein incorporated by reference), a gene which encodes an enzyme forwhich various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a luciferase gene (Ow et al., Science234:856-859 (1986), the entirety of which is herein incorporated byreference); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (USA.)80:1101-1105 (1983), the entirety of which is herein incorporated byreference) which encodes a catechol dioxygenase that can convertchromogenic catechols; an α-amylase gene (Ikatu et al., Bio/Technol.8:241-242 (1990), the entirety of which is herein incorporated byreference); a tyrosinase gene (Katz et al., J. Gen. Microbiol.129:2703-2714 (1983), the entirety of which is herein incorporated byreference) which encodes an enzyme capable of oxidizing tyrosine to DOPAand dopaquinone which in turn condenses to melanin; and anα-galactosidase.

Any of the techniques known in the art for introduction of transgenesinto plants may be used to prepare a plant resistant to fungal diseasein accordance with the invention. Suitable methods for transformation ofplants are believed to include virtually any method by which DNA can beintroduced into a cell, such as by electroporation as illustrated inU.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated inU.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861;and 6,403,865; Agrobacterium-mediated transformation as illustrated inU.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and6,384,301; and protoplast transformation as illustrated in U.S. Pat. No.5,508,184. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells developed into transgenic plants. Techniques useful in thecontext of cotton transformation are disclosed in U.S. Pat. Nos.5,846,797, 5,159,135, 5,004,863, and 6,624,344; and techniques fortransforming Brassica plants in particular are disclosed, for example,in U.S. Pat. No. 5,750,871; and techniques for transforming soybean aredisclosed in for example in Zhang et al. (Plant Cell Tissue Organ Cult56:37-46 (1999) and U.S. Pat. No. 6,384,301.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1: Breeding of Near-Isogenic Lines Containing ASRResistance Loci

One thousand, four hundred single nucleotide polymorphism (SNP) markers,randomly distributed across the 20 linkage groups of the soybean geneticlinkage map, were used to identify SNP markers tightly linked to the ASRresistance locus 1 locus. A panel of soybean lines consisting ofnear-isogenic lines (NILs) developed from a cross between Williams 82and ASR resistance locus 1 donor, PI 200492. Derivative lines of PI200492 were used to identify SNP markers that were polymorphic betweenWilliams 82 and PI 200492. These polymorphic SNP markers were then usedto identify the map location of ASR resistance locus 1 using asegregating backcross population, L85-2378. L85-2378 was developed bycrossing Williams 82 with PI 200492 and five backcross cycles, oressentially 6 doses of Williams 82, were made to recover most ofWilliams 82's desirable traits. Thus L85-2378 is created consisting ofindividuals nearly like the recurrent parent, Williams 82, but eachindividual NIL carries varying amounts or mosaic of genomic regions fromthe donor parent, PI 200492.

The entire population was genotyped with the polymorphic SNP markersidentified above and was subsequently evaluated for soybean rustresistance using a greenhouse assay. Associations between SNP markergenotype and soybean rust resistance phenotype were evaluated. SNPmarkers found to be in high linkage disequilibria with ASR resistancelocus 1 disease phenotypic response were NS0093250, NS0119710,NS0103004, NS0099454, NS0102630, NS0102915, NS0102913, NS0123728,NS0129943, NS0102168, NS0092723, NS0098177, NS0127343, and NS0101121,and are presented in Table 1 and indicated as SEQ ID NOs: 67 through 80.All of these SNP markers map to a region on linkage group G of thepublic soybean genetic linkage map. Table 1 lists sequences for PCRamplification primers, indicated as SEQ ID NOs: 1 through 28, andprobes, indicated as SEQ ID NOs: 100 through 127, corresponding to theseSNP markers. Two SNP markers were identified as being useful inmonitoring the positive introgression of ASR resistance locus 1 andcorrespond to SNP markers NS0102913 and NS0129943 and correspond to SEQID NO: 73 and SEQ ID NO: 75, respectively.

The efficacy of ASR resistance locus 1 against soybean rust isolatesfrom Alabama was also evaluated in the following F2:3 populations:AG4403×PI 200492, AG3302×PI 200492, AG3201×PI 200492, AG26932×PI 200492,AG2402×PI 200492. In each of the populations, a 3:1 segregation ratiowas observed indicating a single dominant gene inheritance pattern.

Following the procedure described for ASR resistance locus 1, the ASRresistance locus 3 locus was mapped using NILs developed from the crossbetween Williams 82 and the donor parent, PI 462312, followed by fivebackcross cycles, or essentially 6 doses of Williams 82, were made torecover most of Williams 82's desirable traits. Thus L85-2378 is createdconsisting of individuals nearly like the recurrent parent, Williams 82but each individual near isogenic line carries varying amounts or mosaicof genomic regions from the donor parent, PI 200492. The entirepopulation was genotyped with the set of polymorphic SNP markersidentified above and was subsequently evaluated for soybean rustresistance using a greenhouse assay. Associations between SNP markergenotype and soybean rust resistance phenotype were evaluated. SNPmarkers found to be in high linkage disequilibria with ASR resistancelocus 3 were NS0099746, NS0123747, NS0126598, NS0128378, NS0096829,NS0125408, NS0098902, NS0099529, NS0097798, NS0137477, NS0095322,NS0136101, and NS0098992, and are presented in Table 1 and indicated asSEQ ID NOs: 81 through 93. These markers were all mapping to LG C2 ofthe public soybean genetic map. Table 1 lists sequences for PCRamplification primers, indicated as SEQ ID NOs: 29 through 54, andprobes, indicated as SEQ ID NOs: 128 through 153, corresponding to theseSNP markers. The marker used to monitor the introgression of ASRresistance locus 3 corresponds to SNP marker N50137477 and is indicatedas SEQ ID NO: 90. To confirm the putative location of ASR resistancelocus 3, a segregating F3:4 population was developed between AVRDC-8 andAG4403. AVRDC-8 is line developed by Asian Vegetable Research andDevelopment Center in Taiwan by crossing Ankur (ASR resistance locus 3containing line) and PI 230970 (ASR resistance locus 2 donor). Thispopulation is currently being genotyped for SNP markers and evaluatedfor resistance reaction against a soybean rust isolate from Loxley, ALto validate the location of ASR resistance locus 3.

The approximate locations of ASR resistance locus 2 and ASR resistancelocus 4 were later determined based upon a survey of polymorphismsbetween a panel of PI lines that are known to contain either ASRresistance locus 2 or ASR resistance locus 4, PI 230970, PI 459025B, thedonor of ASR resistance locus 2 and ASR resistance locus 4,respectively, and other lines that were reported in literature tocontain either QTL. Based on the polymorphism survey, any polymorphicSNP marker is a candidate region near the ASR resistance loci. For ASRresistance locus 2, two candidate regions were identified and the locusis most likely located on linkage group J, near or within the diseaseresistance cluster Brown Stem Rot, Soybean Cyst Nematode resistance andFrog Eye Leaf Spot, or within linkage group N. The ASR resistance locus4 is likely located on linkage group N.

TABLE 1 SNP markers for identification and selection of ASR resistancelocus 1 and ASR resistance locus 3. SEQ ID SEQ ID SEQ ID FORWARD REVERSESEQ ID PROBE MARKER SEQ ID PRIMER PRIMER PROBE 1 2 NS0093250 67 1 2 100101 NS0119710 68 3 4 102 103 NS0103004 69 5 6 104 105 NS0099454 70 7 8106 107 NS0102630 71 9 10 108 109 NS0102915 72 11 12 110 111 NS010291373 13 14 112 113 NS0123728 74 15 16 114 115 NS0129943 75 17 18 116 117NS0102168 76 19 20 118 119 NS0092723 77 21 22 120 121 NS0098177 78 23 24122 123 NS0127343 79 25 26 124 125 NS0101121 80 27 28 126 127 NS009974681 29 30 128 129 NS0123747 82 31 32 130 131 NS0126598 83 33 34 132 133NS0128378 84 35 36 134 135 NS0096829 85 37 38 136 137 NS0125408 86 39 40138 139 NS0098902 87 41 42 140 141 NS0099529 88 43 44 142 143 NS009779889 45 46 144 145 NS0137477 90 47 48 146 147 NS0095322 91 49 50 148 149NS0136101 92 51 52 150 151 NS0098982 93 53 54 152 153

Example 2: Collection and Propagation of Spores

Asian Soybean Rust urediniospores from Phakopsora pachyrhizi werecollected from infected plants at the Monsanto Loxley Agronomy station(Loxley, AL), herein referred to as the Loxley strain.

Soybean plants were inoculated by spraying the underside of the leaveswith spores suspended in water containing 0.01% Tween-20. Lesiondevelopment was visible without magnification at around 7 to 10 dayswith sporulation occurring at 12 to 14 days after infection. Spores fromthe infected plants were collected and resuspended in sterile deionizedwater containing 0.01% Tween 20. The spore concentration was determinedusing a hemacytometer.

Example 3: Detached Leaf Assay for Asian Soybean Rust Resistance

Two types of leaf tissue were assessed for ASR disease phenotyping.Unifoliates leaves, seven to ten days after emergence, or V3 trifoliateleaves, twenty-one to twenty-eight after emergence, were assessed. Atabout two days after emergence from the soil, the soybean plant bears apair of unifoliate leaves which are fully unfurled about five days laterand constitute the first ‘true leaves’. At about seven days afteremergence, the trifoliate leaves appear (comprising three leaves at theend of one petiole). Three sets emerge in sequence and the firsttrifoliate leaves are denoted as the V1 stage, and are fully expanded atten days after emergence. The next two V stages occur a week apart.Notably, the leaves are inoculated for disease after they have bothunfurled and hardened, i.e. not new and green. The unifoliates tend toharden very quickly, around 8-10d after emergence, while V2 and V3trifoliates may not even unfurl completely until up to 24-28 days afteremergence.

Three 3.2 cm diameter Watmann #1 filter papers are placed in each of 6wells of a 6-well tissue culture plate (well volume is 15.5milliliters). The leaves are cut into 3 centimeter by 3 centimeterpieces and placed on top of the Watmann filter papers with the bottom(stomatal side) of the leaf facing upwards. Approximately 2.0milliliters of sterile deionized water is put into each well of the6-well tissue culture plate. Asian Soybean Rust urediniospores fromPhakopsora pachyrhizi are suspended in sterile deionized watercontaining 0.01% tween 20 at a concentration of 1×10⁵ urediniospores permilliliter. Approximately 50 microliters of spore suspension is appliedto each leaf piece using an airbrush (Model Badger 155 Anthem, BadgerAir-Brush Co., Franklin Park, Ill.) with a compressor (Model TC-20,Airbrush Depot, San Diego, Calif.) setting of 1 kilogram per squarecentimeter to wetness. The 6-well plate is then sealed with parafilm andplaced in a growth chamber set to 22 degrees Celsius, with a photoperiodof 12 hours daylength. The plates are checked every 2 or 3 days tomonitor the progression of disease and to assure the wells have notdried out. Deionized water is added to make up the original volume inthe well when needed or incubator relative humidity is adjusted toapproximately 80%. Early symptoms of developing lesions should beevident under a dissecting microscope about 3 to 5 days afterinoculation. Sporulating lesions should be evident 9 to 14 days afterinoculation. Average soybean rust severity scores are calculated frommultiple trials. The rust severity score uses a rating scale from 1 to5; 1—being immune, 2—demonstrating red/brown lesions over less than 50%of the leaf area, 3—demonstrating red/brown lesions over greater than50% of the leaf area, 4—demonstrating tan lesions over less than 50% ofthe leaf area and 5—demonstrating tan lesions over greater than 50% ofthe leaf area. Leaf sections can remain viable in this assay for up to 2months.

Experiments using Asian Soybean Rust susceptible soybean, Lee 74demonstrate consistently high levels of infection for each assayperformed. Further experiments evaluating putative resistant germplasmwere able to differentiate tolerant from susceptible accessions asdemonstrated in Table 2. Accession PI 200487 demonstrated a slow rustresistance phenotype. Efforts are underway to identify markers that willbe used in the introgression of the resistance locus identified in PI200487 into elite germplasm.

In addition, comparison of ASR evaluation of unifoliate and trifoliateleaf tissue showed it takes approximately 45 days from seed to datapoint for trifoliates and approximately 23 days for unifoliates. Bycutting the assay time in half, this significantly economizes thedetached leaf assay and time required to determine disease resistancerating. By saving 3 weeks, plants can be propagated on a faster timescale and susceptible plants can be culled sooner, saving field andgreenhouse space.

TABLE 2 Average rust score for resistant and susceptible accessions asdetermined using unifoliate and trifoliate leaf tissue; Average RustSeverity Score Average Rust Detached Severity Score Accession UnifoliateDetached Leaf Lee 74 5.0 5 PI 200487 1.89 2.25 PI 200492 (ASR resistancelocus 1) 1.00 2 PI 200499 — 5 PI 230970 2.5 3 PI 368038 — 3 PI 368039 —2 PI 462312 — 2 PI 547875 — 2 PI 547878 — 4.25 PI 547879 — 5 Tiana — 5Williams — 5 AVRDC-8 1.8 2.25 Dowling — 5 “—” indicates the assay wasnot performed.

Example 4: Testing of Elite Crosses for Resistance to P. pachyrizi withIntrogressed ASR Resistance Locus 1, ASR Resistance Locus 2 and ASRResistance Locus 3

Crosses with donor resistant parent line, PI 200492, containing ASRresistance locus 1 were performed with various elite lines of soybean tomonitor the positive introgression of ASR resistance locus 1. Leafassays for resistance to the Loxley strain were performed using linesderived from crosses with the resistant parent line accession, PI 200492(ASR resistance locus 1) as well as known resistant accessions (PI230970 (ASR resistance locus 2) and PI 462312 (ASR resistance locus 3))and susceptible elite lines. The resistance scores for all lines testedare presented in Table 3. Average rust severity scores were derived from4 plants, each with 4 replications and rated on 4 different days (10DAI,17DAI, 24DAI, 32DAI).

TABLE 3 Average Rust Severity Score of ASR backcross events and elitelines. Average Progeny Rust From Severity Cross Cross ASR resistancelocus Loci Score Multiple crosses to introgress both ASR AVRDC-8 ASRresistance locus 2/ASR 1.7 resistance locus 2 and ASR resistanceresistance locus 3 locus 3 Known Susceptible Line Dowling Susceptible 5GL_AG4801//L85-2378/L86-1752 JN1137.1 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1753 JN1137.2 ASR resistance locus 1 (MAS) 5GL_AG4801//L85-2378/L86-1754 JN1137.3 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1755 JN1137.4 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1153.1 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1753 JN1153.2 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1754 JN1153.3 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1755 JN1153.4 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1160.1 ASR resistance locus 1 (MAS) 4.8GL_AG4801//L85-2378/L86-1752 JN1160.2 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1160.3 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1160.4 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1163.1 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1163.2 ASR resistance locus 1 (MAS) 4.8GL_AG4801//L85-2378/L86-1752 JN1163.3 ASR resistance locus 1 (MAS) 1GL_AG4801//L85-2378/L86-1752 JN1163.4 ASR resistance locus 1 (MAS) 5GL_AG5501//L85-2378/L86-1752 JN1691.1 ASR resistance locus 1 (MAS) 4.5GL_AG5501//L85-2378/L86-1752 JN1691.2 ASR resistance locus 1 (MAS) 4.6GL_AG5501//L85-2378/L86-1752 JN1691.3 ASR resistance locus 1 (MAS) 4.6GL_AG5501//L85-2378/L86-1752 JN1691.4 ASR resistance locus 1 (MAS) 3GL_AG5501//L85-2378/L86-1752 JN1692.1 ASR resistance locus 1 (MAS) 1.1GL_AG5501//L85-2378/L86-1752 JN1692.2 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1692.3 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1692.4 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1742.1 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1742.2 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1742.3 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1742.4 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1765.1 ASR resistance locus 1 (MAS) 1.2GL_AG5501//L85-2378/L86-1752 JN1765.2 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1765.3 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1765.4 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1774.1 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1774.2 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1774.3 ASR resistance locus 1 (MAS) 1GL_AG5501//L85-2378/L86-1752 JN1774.4 ASR resistance locus 1 (MAS) 1GL_CGL4504D0C//L85-2378/L86-1752 JN1866.1 ASR resistance locus 1 (MAS) 1GL_CGL4504D0C//L85-2378/L86-1752 JN1866.2 ASR resistance locus 1 (MAS) 1GL_CGL4504D0C//L85-2378/L86-1752 JN1866.3 ASR resistance locus 1 (MAS) 1GL_CGL4504D0C//L85-2378/L86-1752 JN1866.4 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2242.1 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2242.2 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2242.3 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2242.4 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2243.1 ASR resistance locus 1 (MAS) 1GL_CGL5400E1X//L85-2378/L86-1752 JN2243.2 ASR resistance locus 1 (MAS)2.4 GL_CGL5400E1X//L85-2378/L86-1752 JN2243.3 ASR resistance locus 1(MAS) 1 GL_CGL5400E1X//L85-2378/L86-1752 JN2243.4 ASR resistance locus 1(MAS) 1.3 GL_CGL5400E1X//L85-2378/L86-1752 JN2250.1 ASR resistance locus1 (MAS) 1 GL_CGL5400E1X//L85-2378/L86-1752 JN2250.2 ASR resistance locus1 (MAS) 1 GL_CGL5400E1X//L85-2378/L86-1752 JN2250.3 ASR resistance locus1 (MAS) 1.2 GL_CGL5400E1X//L85-2378/L86-1752 JN2250.4 ASR resistancelocus 1 (MAS) 1 GL_AG4403//L85-2378/L86-1752 JN774.1 ASR resistancelocus 1 (MAS) 1 GL_AG4403//L85-2378/L86-1752 JN774.2 ASR resistancelocus 1 (MAS) 1.1 GL_AG4403//L85-2378/L86-1752 JN774.3 ASR resistancelocus 1 (MAS) 1 GL_AG4403//L85-2378/L86-1752 JN774.4 ASR resistancelocus 1 (MAS) 1

Lines containing the ASR resistance locus 1 locus showed greatestresistance to the Loxley strain. Introgression of the ASR resistancelocus 1 was confirmed by MAS.

Example 5: Testing of Soybean Accessions for ASR Resistance Using theDetached Leaf Assay

Seven hundred putative ASR resistant accessions were identified basedupon greenhouse assays, using a mixed population of ASR isolates offoreign origin. Leaf to assays for resistance to ASR were performed asdescribed in Example 3 using a subset of two hundred and fifty of theseven hundred USDA putative resistant accessions. A complementary set oftwo hundred and fifty ASR susceptible accessions from the USDA wereselected for comparison in the leaf assay based upon matching maturitiesand geographic origins to the two hundred and fifty resistantaccessions. The average rust severity scores of the most resistantaccessions (those exhibiting an average rust severity score from 1 to 2)is presented in Table 4 below. One thousand, four hundred SNP markers,distributed every 5 centimorgans across the 20 linkage groups of thesoybean genetic linkage map, will be used to identify markers useful infollowing the introgression of the ASR resistance loci possessed by theresistant accessions into elite germplasm.

TABLE 4 Average Rust Severity Score ASR Resistant Accessions. AverageRust Severity Accession Score PI200488 1.0 PI200492 1.0 PI203398 1.0PI307884B 1.0 PI416764 1.0 PI416826A 1.0 PI417117 1.0 PI417132 1.0PI423967 1.0 PI506947 1.0 PI507009 1.0 PI507259 1.0 PI561305 1.0PI567031B 1.0 PI567034 1.0 PI567056A 1.0 PI567058D 1.0 PI567190 1.0PI605773 1.0 PI605829 1.0 PI605865B 1.0 PI379620 1.3 PI416873B 1.3PI417128 1.3 PI417463 1.3 PI567123A 1.3 PI578457A 1.3 PI615437 1.3PI379621 1.3 PI567102B 1.3 PI594172A 1.3 PI628932 1.3 PI079648 1.5PI291309C 1.5 PI416886 1.5 PI417503 1.5 PI506491 1.5 PI506677 1.5PI506695 1.5 PI507193 1.5 PI567046A 1.5 PI567053 1.5 PI567189A 1.5PI605891B 1.5 PI200455 1.8 PI232989 1.8 PI594494A 1.8 PI597405D 1.8PI069533 2.0 PI084674 2.0 PI230970 2.0 PI291278 2.0 PI341252 2.0PI417126 2.0 PI417134 2.0 PI417208 2.0 PI423923 2.0 PI437609A 2.0PI471900 2.0 PI497969 2.0 PI506628 2.0 PI547875 2.0 PI567024 2.0PI567025A 2.0 PI578471A 2.0 PI594512C 2.0 PI594561 2.0 PI605781A 2.0PI605838 2.0 PI606405 2.0 PI606440A 2.0 PI615445 2.0

In addition, SNP markers distributed proximal and distal to ASRresistance locus 3 were genotyped for a set of eighty-nine resistantaccessions. Four additional SNP markers (NS0103749, NS0118897,NS0119715, and NS0130920) were found to be associated with ASRresistance locus 3 and are listed in Table and indicated as SEQ ID NOs:94 through 97. Table 5 lists sequences for PCR amplification primers,indicated as SEQ ID NOs: 55 through 62, and probes, indicated as SEQ IDNOs: 154 through 161, corresponding to these SNP markers.

This information will be used to identify novel resistance sourcesuseful in to prioritizing the introgression of the ASR and otherpathogen resistance loci.

TABLE 5 SNP markers for identification and selection of ASR resistancelocus 3. SEQ ID SEQ ID SEQ ID FORWARD REVERSE SEQ ID PROBE MARKER SEQ IDPRIMER PRIMER PROBE 1 2 NS0103749 94 55 56 154 155 NS0118897 95 57 58156 157 NS0119715 96 59 60 158 159 NS0130920 97 61 62 160 161

Example 6. Using Association Studies to Identify QTL that Confer FungalDisease Resistance

To identify regions or genes associated with the disease is the firststep toward developing resistant varieties. Four loci for rustresistance (ASR resistance locus 1, ASR resistance locus 2, ASRresistance locus 3, ASR resistance locus 4) were previously identified.In this example, linkage disequilibrium and haplotype associationmapping were applied to a case-control data sample from soybeangermplasm.

Four hundred ninety-two soybean lines (246 resistant-susceptible pairs)were scored for rust resistance as well as fingerprinted using 797 SNPs.Disease resistance was scored in 1 to 5 scales to a mixture ofPhakopsora pachyrhizi isolates, with less than 3 as resistant andgreater than 4 as susceptible. Specifically, case-control testing,Fishers' exact test, single marker F-test, and haplotype trendregression were explored on window sizes of 3, 5 and 9 consecutive SNPs.Multiple testing results significantly associate two SNP markers fromtwo separate haplotype windows, referred to herein as in fungal diseaseresistance haplotype windows 1 and 2, on chromosome 13 24-45 cM) withresistance to fungal disease. The SNP markers NS0103033 and NS0124935are located in fungal disease resistance haplotype windows 1 and 2respectively. The primers for NS0103033 (SEQ ID NO: 98) are indicated inSEQ ID NOs: 63 and 64 and the probes are indicated in SEQ ID NOs: 162and 163. The primers for NS0124935 (SEQ ID NO: 99) are indicated in SEQID NOs: 65 and 66 and the probes are indicated in SEQ ID NOs: 164 and165. Resistance scores for each of the haplotypes and the marker allelefor each haplotype are indicated in Table 6. Each window is designatedby five SNP markers and the alleles for each is indicated as haplotypesequence. The allele for NS0103033 in haplotype window 1 and NS0124935in haplotype window 2 are indicated in bold. For NS0103033, the SNP isactually a 9-bp indel where “Z” represents the deletion (*********) and“W” represents the insertion (GAAGTGGAT).

Varieties containing resistant haplotypes from haplotype window 1 and/or2 are indicated in table 7. This mapping effort has identifiedadditional ASR disease resistance QTLs in addition to the previouslydefined ASR resistance loci.

TABLE 6 Summary scoring for lines containing resistanthaplotypes in ASR resistance haplotype windows 1 and 2. A resistance score of 0 indicates theline was resistant and a score of 1 indicates theline was designated susceptibe. ASR Resistance resistance HaplotypeHaplotype Score locus Window 1 sequence  0 1 5 Haplotype 1 AAZA?  5  0 6Haplotype 2 AGWGA 26 10 7 Haplotype 3 AGWGG 34 15 8 Haplotype 4 TAZAG  5 0 9 Haplotype 5 TAZGA 13  5 ASR Resistance resistance HaplotypeHaplotype Score locus Window 2 sequence  0  1 10 Haplotype 6 CGTTG  8  111 Haplotype 7 GGTTC 26 11 12 Haplotype 8 GGCCC 12  6 13 Haplotype9GGT-C  4  0

TABLE 7 Disease ratings for resistant germplasm containing haplotypes inASR resistance windows 1 and/or 2 on chromosome 13. ResistanceResistance haplotype from haplotype from haplotype haplotype Line Ratingwindow 1 window 2 PI164885 2.5 X X PI165524 2 X X PI166028 2 X PI1899682 X X PI200446 2 X PI200488 2.5 X PI205901B 2.5 X PI222549 2.5 XPI224270 2.5 X PI227331 2.5 X X PI229333 2.5 X PI238109 2.3 X PI240667A1 X PI258383 2 X PI291309C 2 X PI341252 2.5 X X PI374189 2.3 X PI3983352 X PI399070 2.5 X PI407831 2.5 X PI407833C 2 X PI407845A 2.5 X PI4078582.3 X X PI407881 2.3 X PI408088 2.3 X PI408134B 2 X PI408272B 2 XPI417122 2.5 X PI417126 2.5 X PI417235 2 X PI417335 2.3 X PI423717 2 XPI423722 2.3 X PI423730B 2.3 X PI423852 2.3 X X PI424190 2.5 X PI434973A2.5 X PI437110A 2.3 X PI437437A 1.5 X PI437740B 2.3 X X PI437921 2 XPI437982 2.3 X X PI438073 2.3 X PI438371 2.5 X PI438480 2.5 X PI4797352.3 X PI497965 2.5 X PI506737 2 X PI506863 2 X PI507142 2.5 X PI508269 2X PI548325 2 X PI561289 2 X X PI561329 2.5 X PI561330A 2 X PI561337 2 XPI561377 2.3 X PI566978 2.5 X PI567010B 2.3 X PI567093B 2 X X PI567104B2.5 X X PI567108B 2.5 X X PI567129 2.3 X X PI567140B 2.5 X PI567174C 2.3X PI567175C 2 X X PI567300A 2 X PI567409A 2.3 X PI567470 2 X PI567473C2.5 X PI567474 2.3 X PI567489A 2 X PI567507B 2 X PI567554A 2 X PI5675602.5 X X PI567561 2.5 X PI567675 2.3 X PI567692 2 X X PI567718 2 X XPI567780A 2.3 X PI578305B 2.5 X PI587598A 2.5 X PI587914B 2 X PI587922A2 X PI587935A 2.3 X PI588000 2.5 X PI588034 2.5 X PI592962B 2.3 XPI594525 2.5 X X PI594538A 2 X X PI594767B 1 X PI597480A 2.3 X PI603293B2.3 X PI603296 2.5 X PI603429D 2.5 X PI603564A 2.3 X PI603612 2.3 X XPI603704A 2.5 X X PI605891B 2.5 X PI628870 1.5 X PI628932 2.4 X

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A method for assaying a soybean plant for disease resistance,immunity, or susceptibility comprising the steps of: a. detaching aplant tissue from said soybean plant; b. cultivating said tissue in amedia; c. exposing said tissue to a plant pathogen; and d. assessingsaid tissue for resistance, immunity, or susceptibility to diseasecaused by said pathogen. 2.-26. (canceled)